Mesothelin vaccines and model systems

ABSTRACT

Mesothelin can be used as an immunotherapeutic target. It induces a cytolytic T cell response. Portions of mesothelin which induce such responses are identified. Vaccines can be either polynucleotide- or polypeptide-based. Carriers for raising a cytolytic T cell response include bacteria and viruses. A mouse model for testing vaccines and other anti-tumor therapeutics and prophylactics comprises a strongly mesothelin-expressing, transformed peritoneal cell line.

The contents of each of the following applications are specificallyincorporated herein: this is a continuation application of Ser. No.10/618,088 filed Jul. 14, 2003, which is a provisional U.S. ApplicationSer. Nos. 60/395,556, filed Jul. 12, 2002, 60/398,217, filed Jul. 24,2002, Ser. No. 60/414,931, filed Sep. 30, 2002, and Ser. No. 60/475,783filed Jun. 5, 2003.

This invention was made using funds from the U.S. government. The termsof grants NCI CA62924, NCI RO1 CA72631, NCI RO1 CA71806, U19 CA72108-02,and NCDDG RFA CA-95-020 mandate that the U.S. government retain certainrights in the invention.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

The invention relates to the field of cancer therapeutics, cancerprognosis, and anti-cancer drug development. In some aspects it relatesto mesothelin as a therapeutic target. In another aspect it relates todeveloping other therapeutic targets.

BACKGROUND OF THE INVENTION

Transformation from a normal to a malignant cell involves complexgenetic and epigenetic changes, affecting a large number of genes (1,2). Many of these altered genes are translated into new, altered, oroverexpressed proteins that may represent candidate targets for immunerejection. T cell screening of cDNA libraries isolated from tumor cells,biochemical elution and purification of major histocompatibility complex(MHC) bound antigens, and antibody screening of phage display libraries(SEREX method) have greatly facilitated the identification of tumorantigens, particularly those expressed by malignant melanomas (3-13). Asa result, there are a number of antigen-specific vaccine approachesunder clinical development for this disease (3-6, 14). Unfortunately,these antigen identification approaches have not been successful foridentifying antigens expressed by many other common cancers. The majorlimitation has been the inability to generate patient-derived T celllines and clones that can be employed to identify immune relevant tumortargets. Furthermore, T cell responses to specific human tumor antigenshave not yet been correlated with clinical responses afterimmunotherapy.

The recent development of high throughput technologies that can quantifygene expression in human tissues has led to the identification of alarge number of genes that are differentially expressed in tumorsrelative to the normal tissue from which they derive (15-18). These geneexpression databases can be used as initial filters upon which to applya functional immune-based screening strategy (19). A growing number ofgenes shown to be differentially expressed in pancreatic adenocarcinomasusing serial analysis of gene expression (SAGE) have been tabulated andreported (20-22). However, it is unclear which of these differentiallyexpressed genes are immunologically relevant for an anti-tumor response.There is a need in the art for a way of identifying immunologicallyrelevant proteins among the proteins which are differentially expressedin tumor and normal tissues.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment a method is provided for inducing a T-cellresponse to a tumor that overexpresses mesothelin relative to normaltissue from which the tumor is derived. The tumor can be, for example,an ovarian cancer, a pancreatic cancer, a mesothelioma, or a squamouscell carcinoma. A vaccine comprising a polypeptide comprising an MHCClass I- or Class II-binding epitope of mesothelin is administered to apatient who has said tumor or who has had said tumor removed. Thepatient can also be one who is at risk of developing such a tumor. Theepitope binds to an allelic form of MHC class I or MHC class II which isexpressed by the patient. A T-cell response to mesothelin is therebyinduced. The vaccine does not comprise whole tumor cells. Thepolypeptide is optionally mesothelin. The T-cell response may be a CD4⁺T-cell response and/or a CD8⁺ T-cell response.

In a second embodiment a method is provided for inducing a T-cellresponse to a tumor that overexpresses mesothelin relative to normaltissue from which the tumor is derived. The tumor can be, for example,an ovarian cancer, a pancreatic cancer, a mesothelioma, or a squamouscell carcinoma. A vaccine comprising a polynucleotide encoding apolypeptide comprising an MHC Class I- or MHC Class II-binding epitopeof mesothelin is administered to a patient who has said tumor or who hashad said tumor removed. The patient can also be one who is at risk ofdeveloping such a tumor. The epitope binds to an allelic form of MHCclass I or class II which is expressed by the patient. A T-cell responseto mesothelin is thereby induced. The vaccine does not comprise wholetumor cells. The polypeptide encoded by the polynucleotide of thevaccine is optionally mesothelin. The T-cell response may be a CD4⁺T-cell response and/or a CD8⁺ T-cell response.

In a third embodiment a method is provided for identifying immunogensuseful as candidates for anti-tumor vaccines. A protein is selectedwhich is expressed by a tumor and which is minimally or not expressed bynormal tissue from which the tumor is derived. Preferably the protein isexpressed by a greater than 10% of tumor isolates tested of a type oftumor. Lymphocytes of humans who have been vaccinated with a vaccinewhich expresses the protein are tested to determine if the lymphocytescomprise CD8⁺ T cells or CD4⁺ T cells which are specific for theprotein. The presence of the CD8⁺ T cells or CD4⁺ T cells indicates thatthe protein is a candidate for use as an anti-tumor vaccine.

A fourth embodiment of the invention provides a method of predictingfuture response to a tumor vaccine in a patient who has received thetumor vaccine. Lymphocytes of the patient are tested to determine if thelymphocytes comprise CD8⁺ T cells or CD4⁺ T cells which are specific foran antigen in the vaccine. The presence of said CD8⁺ T cells or CD4⁺ Tcells predicts a longer survival time than the absence of said CD8⁺ Tcells or CD4⁺ T cells.

A fifth embodiment of the invention provides a vaccine which induces aCD8⁺ T cell or CD4⁺ T cell response. The vaccine comprises a polypeptidecomprising an MHC Class I- or MHC Class II-binding epitope ofmesothelin. The epitope binds to an allelic form of MHC class I or classII which is expressed by the patient. A T-cell response to mesothelin isthereby induced. The vaccine does not comprise whole tumor cells. Thevaccine further comprises a carrier for stimulating a T cell immuneresponse. The polypeptide is optionally mesothelin.

Another embodiment of the invention provides another vaccine whichinduces a CD8⁺ T cell or CD4⁺ T cell response. The vaccine comprises apolynucleotide encoding a polypeptide comprising an MHC Class I- or MHCClass II-binding epitope of mesothelin. The epitope binds to an allelicform of MHC class I or class II which is expressed by the patient. ACD8⁺ T cell or CD4⁺ T cell response to mesothelin is thereby induced.The vaccine does not comprise whole tumor cells. The vaccine furthercomprises a carrier for stimulating a T cell immune response. Thepolypeptide encoded by the polynucleotide of the vaccine is optionallymesothelin.

Another embodiment of the invention provides an isolated polypeptide of9 to 25 amino acid residues. The polypeptide comprises an epitopeselected from the group consisting of SLLFLLFSL (SEQ ID NO: 1);VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY (SEQ IDNO: 4); FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).

Yet another embodiment of the invention provides an antibody that bindsto an epitope selected from the group consisting of SLLFLLFSL (SEQ IDNO: 1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ ID NO: 3); ALQGGGPPY(SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); and LYPKARLAF (SEQ ID NO: 6).

Yet another embodiment of the invention provides a CD8⁺ T cell or CD4⁺ Tcell line that binds to MHC class I-peptide complexes, wherein thepeptide comprises an epitope selected from the group consisting ofSLLFLLFSL (SEQ ID NO: 1); VLPLTVAEV (SEQ ID NO: 2); ELAVALAQK (SEQ IDNO: 3); ALQGGGPPY (SEQ ID NO: 4); FYPGYLCSL (SEQ ID NO: 5); andLYPKARLAF (SEQ ID NO: 6).

A tenth embodiment of the invention provides a method for predictingfuture response to a tumor vaccine in a patient who has received thevaccine. The tumor vaccine comprises at least one T-cell epitope ofmesothelin. The patient is tested to determine if the patient has adelayed type hypersensitivity (DTH) response to mesothelin, wherein thepresence of said response predicts a longer survival time than theabsence of said response.

An eleventh embodiment of the invention provides a recombinant mousecell line which comprises peritoneal cells which have been transformedby HPV-16 genes E6 and E7 and an activated oncogene. The cell line iscapable of forming ascites and tumors upon intraperitoneal injectioninto an immunocompetent mouse.

Also provided is a mouse model which comprises a mouse which has beeninjected with a recombinant mouse cell line. The recombinant mouse cellline comprises peritoneal cells transfected by HPV-16 genes E6 and E7and an activated oncogene. The former genes immortalize and the lattergene transforms. The cell line is capable of forming ascites and tumorsupon intraperitoneal injection into an immunocompetent mouse.

Another aspect of the invention is a method of testing a substance todetermine if it is a potential drug for treating a cancer. The cancermay be, for example, an ovarian cancer, a pancreatic cancer, amesothelioma, or a squamous cell carcinoma. A test substance iscontacted with a mouse model. The mouse model comprises a mouse that hasbeen injected with a recombinant mouse cell line. The injection can beaccomplished before or after the test substance is contacted with themouse. The recombinant mouse cell line comprises peritoneal cells whichhave been transfected by HPV-16 genes E6 and E7 and an activatedoncogene. The cell line is capable of forming ascites and tumors uponintraperitoneal injection into an immunocompetent mouse. One determineswhether the test substance causes delay of tumor formation or regressionof a tumor in the mouse model, diminution of ascites volume in the mousemodel, or longer survival time in the mouse model. Any of these effectsindicates that the test substance is a potential drug for treatingcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1F show a T2 binding assay that identifies mesothelin andPSCA protein derived epitopes that bind to HLA-A2, A3, and A24molecules. T2 cells were pulsed with 100-400 micrograms of peptideovernight at room temperature before analysis by flow cytometry. FIG.1A. T2 cells expressing HLA-A2 and pulsed with either: no peptide (leftpeak), a Mesothelin A1309-318 binding peptide (left peak), MesothelinA220-29 (right peak), and Mesothelin A2530-539 (middle peak). Peptidepulsed cells were stained with an unlabeled mouse anti-HLA class Imolecule monoclonal antibody W6/32 and a goat-anti-mouse FITC-labeledIgG2a secondary antibody. FIG. 1B. T2 cells genetically modified toexpress A3 and pulsed with either: no peptide (left peak), MesothelinA1309-318 binding peptide (left peak), Mesothelin A383-92 (middle peak),and Mesothelin A3225-234 (right peak). Peptide pulsed cells were stainedwith an unlabeled mouse anti-human HLA-A3 specific monoclonal antibodyGAPA3 and a FITC-labeled IgG2a secondary antibody. FIG. 1C. T2 cellsgenetically modified to express A24 and pulsed with either: no peptide(left peak), Mesothelin A1309-318 peptide (second from left peak),Mesothelin A24435-444 (right peak), and Mesothelin A24475-484 (thirdfrom left peak). Peptide pulsed cells were stained with an unlabeledpan-HLA antibody W6/32 and a FITC-labeled IgG2a secondary antibody. FIG.1D. T2 cells expressing HLA-A2 and pulsed with either: MesothelinA1309-318 binding peptide (left peak), PSCA A25-13 (fourth from leftpeak), PSCA A214-22 (second from left peak), PSCA A2108-116 (third fromleft peak) and PSCA A243-51 (right peak). Peptide pulsed cells werestained with an unlabeled mouse anti-HLA class I molecule monoclonalantibody W6/32 and a goat-anti-mouse FITC-labeled IgG2a secondaryantibody. FIG. 1E. T2 cells genetically modified to express A3 andpulsed with either: Mesothelin A1309-318 binding peptide (left peak),PSCA A399-107 (fourth from left peak), A35-13 (second from left peak),A314-22 (third from left peak), A3109-117 (right peak), A343-51 (fifthfrom left peak), and PSCA A320-28 (sixth from left peak). Peptide pulsedcells were stained with an unlabeled mouse anti-human HLA-A3 specificmonoclonal antibody GAPA3 and a FITC-labeled IgG2a secondary antibody.FIG. 1F. T2 cells genetically modified to express A24 and pulsed witheither: Mesothelin A1309-318 peptide (right peak), PSCA A2476-84 (rightpeak), PSCA A24108-116 (third from left peak), PSCA A2499-107 (rightpeak), PSCA A24109-117 (second from left peak), and PSCA A2477-85(second from left peak). Peptide pulsed cells were stained with anunlabeled pan-HLA antibody W6/32 and a FITC-labeled IgG2a secondaryantibody.

FIGS. 2A to 2D shows an ELISPOT analysis of CD8+ T cells from PBMCswhich demonstrates post-vaccination induction of mesothelin-specific Tcells in three DTH responders but not in 11 non-DTH responders whoreceived an allogeneic GM-CSF-secreting tumor vaccine for pancreaticcancer. FIG. 2A. ELISPOT analysis of PBL from two patients who wereHLA-A3 positive; FIG. 2B. ELISPOT analysis of PBL from two patients whowere HLA-A 2 and HLA-A3 positive; FIG. 2C. ELISPOT analysis of PBL fromtwo patients who were HLA-A24 positive. FIG. 2D. ELISPOT analysis wasperformed on PBL from all 14 patients who were treated on the phase Iallogeneic GM-CSF secreting pancreatic tumor vaccine study (28). ELISPOTanalysis for IFN-γ-expressing cells was performed using PBMC that wereisolated on the day prior to vaccination or 28 days following the firstvaccination. Lymphocytes were isolated by ficoll-hypaque separation andstored frozen in liquid nitrogen until the day of assay. CD8+ T cellenrichment was performed prior to analysis. T2-A3 cells were pulsed withthe two mesothelin derived epitopes MesoA3(83-92) (open squares),MesoA3(225-234) (closed circle) and HIV-NEFA3 (94-103) (open triangle).T2-A2 cells were pulsed with the two mesothelin derived epitopesMesoA2(20-29) (closed squares), MesoA2(530-539) (open circle), andHIV-GAG(77-85), (closed triangle). T2-A24 cells were pulsed with the twomesothelin derived epitopes MesoA24 (435-444) (open diamond),MesoA24(475-484) (closed diamond), and tyrosinase A24(206-214) (star).All DTH responders are represented by the three lower lines, and DTHnon-responders are represented by the nine upper lines. For thedetection of nonspecific background, the number of IFN-γ spots for CD8+T cells specific for the irrelevant control peptides were counted. TheHLA-A2 binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ ID NO:7),the HLA-A3 binding HIV-NEF protein derived epitope (QVPLRPMTYK; SEQ IDNO: 8), and the HLA-A24 binding tyrosinase protein derived epitope(AFLPWHRLF; SEQ ID NO: 9) were used as negative control peptides inthese assays. Data represents the average of each condition assayed intriplicate and standard deviations were less than 5%. Plotted are the #of human interferon gamma (hIFNg) spots per 10⁵ CD8+ T cells. Analysisof each patient's PBL was performed at least twice.

FIG. 3 shows an ELISPOT analysis performed to assess the recognition ofthe influenza matrix protein HLA-A2 binding epitope M1 (GILGFVFTL; SEQID NO: 10) on PBL from all 5 patients on the study who were HLA-A2positive (4 non-DTH responders and 1 DTH responder). This analysis wasperformed on the same PBL samples described for FIGS. 2A to 2D above.The DTH responders are represented by lower lines, and the DTHnon-responders are represented by upper lines. For the detection ofnonspecific background, the number of IFN-γ spots for CD8+ T cellsspecific for the irrelevant control peptides were counted. The HLA-A2binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ ID NO: 7), theHLA-A3 binding HIV-NEF protein derived epitope (QVPLRPMTYK; SEQ ID NO:8), and the HLA-A24 binding melanoma tyrosinase protein derived epitope(AFLPWHRLF; SEQ ID NO: 9) were used as negative control peptides inthese assays. Data represents the average of each condition assayed intriplicate and standard deviations were less than 5%. Plotted are the #of human interferon gamma (hIFNg) spots per 10⁵ CD8+ T cells. Analysisof each patient's PBL was performed at least twice and all ELISPOTassays were performed in a blinded fashion.

FIGS. 4A to 4D shows an ELISPOT analysis of CD8+ T cells from PBMCs. Nopost-vaccination induction was observed of PSCA-specific T cells in DTHresponders or non-DTH responders who received an allogeneicGM-CSF-secreting tumor vaccine for pancreatic cancer. FIG. 4A. ELISPOTanalysis of PBL from two patients who were HLA-A3 positive; FIG. 4B.ELISPOT analysis of PBL from two patients who were HLA-A 2 and HLA-A3positive; FIG. 4C. ELISPOT analysis of PBL from two patients who wereHLA-A24 positive. FIG. 4D. ELISPOT analysis of PBL from eight patientswho were non-responders. ELISPOT analysis for IFN-γ-expressing cells wasperformed using PBMC that were isolated on the day prior to vaccinationor 28 days following each of the vaccination. Lymphocytes were isolatedby ficoll-hypaque separation and stored frozen in liquid nitrogen untilthe day of assay. CD8+ T cell enrichment was performed prior toanalysis. T2-A3 cells were pulsed with the six PSCA derived epitopes:PSCAA3(7-15) (closed squares), PSCAA3(52-60) (closed diamond),PSCAA3(109-117) (closed triangle), PSCAA3(43-51) (open square),PSCAA3(20-28) (open diamond), and PSCAA3(99-107) (open triangle).Negative HIV-NEFA3 (94-103) values were subtracted out. T2-A2 cells werepulsed with the three PSCA derived epitopes: PSCAA2(5-13) (closedsquares), PSCAA2(14-22) (closed diamonds), PSCAA2(108-116) (closedtriangles). Negative HIV-GAG(77-85) values were subtracted out. T2-A24cells were pulsed with the five PSCA derived epitopes: PSCAA24(76-84)(closed diamond), PSCAA24(77-85) (star), PSCAA24(109-117) (closedtriangles), PSCAA24(108-116) (closed circle), and PSCAA24(99-107) (opentriangle). Negative Tyrosinase A24(206-214) values were subtracted. Forthe detection of nonspecific background, the number of IFN-γ spots forCD8+ T cells specific for the irrelevant control peptides were counted.The HLA-A2 binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ IDNO: 7), the HLA-A3 binding HIV-NEF protein derived epitope (QVPLRPMTYK;SEQ ID NO: 8), and the HLA-A24 binding tyrosinase protein derivedepitope (AFLPWHRLF; SEQ ID NO: 9) were used as negative control peptidesin these assays. Data represents the average of each condition assayedin triplicate and standard deviations were less than 5%. The number ofhuman interferon gamma (hIFNg) spots per 105 CD8+ T cells is plotted.Analysis of each patient's PBL was performed at least twice.

FIG. 5 shows expression of surface Mesothelin and PSCA on Panc 6.03 andPanc 10.05 vaccine lines. The pancreatic tumor vaccine lines Panc 6.03(top two panels) and Panc 10.05 (bottom two panels) were analyzed byflow cytometry for their levels of surface mesothelin and PSCA using themesothelin specific monoclonal antibody CAK1 (left panels) and the PSCAspecific monoclonal antibody 1G8 (right panels) as the primary antibodyand goat anti-mouse IgG FITC as the secondary antibody. The solid linerepresents the isotype control, the shaded area (left panels) representsmesothelin staining, and the shaded area fright panels) represents PSCAstaining.

FIGS. 6A to 6C show that mesothelin-specific CD8+ T cells are detectedfollowing multiple vaccinations with an allogeneic GM-CSF secretingtumor vaccine in DTH-responders but not in non-DTH responders. FIG. 6A.ELISPOT analysis of PBL from two patients who were HLA-A3 positive; FIG.6B. ELISPOT analysis of PBL from two patients who were HLA-A 2 andHLA-A3 positive; FIG. 6C. ELISPOT analysis of PBL from two patients whowere HLA-A24 positive. ELISPOT analysis for IFN-γ-expressing cells wasperformed using PBMC that were isolated on the day prior to vaccinationor 28 days following each vaccination as described in FIGS. 2A to 2D.Each peptide has the same symbol code as described for FIGS. 2A to 2D.The DTH responders are represented by the upper lines and the DTHnon-responders are represented by the lower lines. For the detection ofnonspecific background, the number of IFN-γ spots for CD8+ T cellsspecific for the irrelevant control peptides were counted. The HLA-A2binding HIV-GAG protein derived epitope (SLYNTVATL; SEQ ID NO: 7), theHLA-A3 binding HIV-NEF protein derived epitope (QVPLRPMTYK; SEQ ID NO:8), and the HLA-A24 binding melanoma tyrosinase protein derived epitope(AFLPWHRLF; SEQ ID NO: 9) were used as negative control peptides inthese assays. Data represent the average of each condition assayed intriplicate and standard deviations were less than 5%. Plotted are thenumber of human interferon gamma (hIFNg) spots per 105 CD8+ T cells.Analysis of each patient's PBL was performed at least twice.

FIGS. 7A to 7C show the generation and characterization of anascitogenic ovarian tumor cell line (WF-3) in mice. WF-3 tumor cellswere injected into C57BL/6 mice intraperitoneally at a dose of 1×10⁵cells/mouse. Mice were euthanized 4 weeks after tumor challenge (7A)Representative gross picture to demonstrate ascites formation in mice.Note: Mice developed significant ascites with an increase in abdominalgirth 4 weeks after tumor challenge. (7B) Hematoxylin and eosin stainingof the explanted tumors viewed at 90× magnification. The tumorsdisplayed a papillary configuration, morphologically consistent withtumors derived from the peritoneum or ovaries. Tumors viewed at 400×magnification. The inset displays the features of a WF-3 tumor cell ingreater detail.

FIGS. 8A and 8B show MHC class I (FIG. 8A) and MHC class II (FIG. 8B)presentation on the mouse WF-3 tumor cells. WF-3 tumor cells wereharvested, trypsinized, washed, and resuspended in FACSCAN buffer.Anti-H2 Kb/H-2D monoclonal antibody or anti-I-Ab monoclonal antibody wasadded, followed by flow cytometry analysis to detect MHC class I andclass II expression on WF-3 tumor cells. (8A) WF-3 tumor cells werepositive for MHC class I presentation (thick line) compared to the MHCclass I-negative control (thin line). (8B) WF-3 tumor cells werenegative for MHC class II presentation. The thin line indicates stainingof the MHC class II-negative control.

FIGS. 9A to 9B show the effect of WF-3 tumor dose on ascites formationin two independent trials shown in FIG. 9A and FIG. 3B. WF-3 tumor cellswere injected into C57BL/6 mice intraperitoneally at various doses(1×10⁴, 5×10⁴, 1×10⁵ and 1×10⁶ cells/mouse). Mice were monitored twice aweek for ascites formation and tumor growth. Note: All of the miceinjected with 5×10⁴, 1×10⁵ and 1×10⁶ cells intraperitoneally, developedascites and tumor growth within 30 days. 20% of mice injected with 1×10⁴cells were tumor-free without ascites formation after 90 days of tumorinjection. The data are from one representative experiment of twoperformed.

FIG. 10 shows expression of murine mesothelin in WF-3 tumor cellsdemonstrated by RT-PCR with gel electrophoresis. FIG. 10. RT-PCR. RT-PCRwas performed using the Superscript One-Step.RT-PCR Kit (Gibco, BRL) anda set of primers: 5′-CCCGAATTCATGOCCTTGCCAACAGCTCGA-3′ (SEQ ID NO: 11)and 5′-TATGAATCCGCTCAGCCTTAAAGCTGGGAG-3′ (SEQ ID NO: 12). Lane 1, sizemarker. Lane 2, RNA from W-3 cells and Lane 3, RNA frommesothelin-negative B 16 tumor cells. Specific amplification (indicatedby an arrow) was observed in Lane 2 (WF-3 cells) but not in the Lane 3(B 16 cells).

FIG. 11 shows in vivo tumor protection experiments against WF-3 tumorgrowth using mesothelin-specific DNA vaccines. Mice received a boosterwith the same dose one week later, followed by intraperitoneal challengewith 5×10⁴ WF-3 cells/mouse one week afterward. Ascites, formation inmice was monitored by palpation and inspection. Mice were, sacrificed atday 90. Note: Vaccination with pcDNA3-mesothelin DNA resulted in asignificantly higher percentage of tumor-free mice than vaccination withother DNA. (P<0.001). Results shown here are from one representativeexperiment of two performed.

FIG. 12 shows CTL assays which demonstrate specific lysis induced byvaccination with mesothelin-specific DNA vaccines. Mice (5 per group)were immunized with various DNA vaccines intradermally. Mice received abooster with the same dose one week later. Splenocytes from mice werepooled 14 days after vaccination. To perform the cytotoxicity assay,splenocytes were cultured with mesothelin protein—for 6 days and used aseffector cells. WF-3 tumor cells served as target cells. WF-3 cells weremixed with splenocytes at various E:T ratios. Cytolysis was determinedby quantitative measurements of LDH. Note: The pcDNA3-mesothelin DNAvaccine generated a significantly higher percentage of specific lysisthan the other DNA vaccines (P<0.001). The data presented in this figureare from one representative experiment of two performed.

DETAILED DESCRIPTION OF THE INVENTION

The recent development of high-throughput technologies that quantifygene expression has led to the identification of many genes that aredifferentially expressed in human cancers. However, differentialexpression does not, on its own, indicate that an antigen is atherapeutic target. Therefore, a functional immunologic screen wasapplied to a SAGE gene expression database in order to identifyimmunologically relevant tumor antigens. We previously reported theassociation of prolonged disease-free survival and in vivo induction ofanti-tumor immunity in three of fourteen patients receiving a pancreatictumor vaccine. Here we identify mesothelin as a tumor antigen recognizedby uncultured CD8+ T cells isolated from these vaccinated patients.Moreover, the induction of mesothelin-specific T cells was not found inthe eleven other patients who received the same vaccine but relapsed. Tovalidate mesothelin as a tumor antigen, we show that none of thepatients respond to another differentially expressed gene product,prostate stem cell antigen. These data identify mesothelin as an invitro marker of vaccine induced immune responses that correlate withclinical anticancer responses. The inventors also describe a functionalgenomic approach for identifying and validating other immunologicallyrelevant human tumor antigens.

The vaccines of the present invention can be administered by any meansknown in the art for inducing a T cell cytolytic response. These meansinclude oral administration, intravenous injection, percutaneousscarification, subcutaneous injection, intramuscular injection, andintranasal administration. The vaccines can be administeredintradermally by gene gun. Gold particles coated with DNA may be used inthe gene gun. Other inoculation routes as are known in the art can beused.

Additional agents which are beneficial to raising a cytolytic T cellresponse may be used as well. Such agents are termed herein carriers.These include, without limitation, B7 costimulatory molecule,interleukin-2, interferon-γ, GM-CSF, CTLA-4 antagonists, OX-40/OX-40ligand, CD40/CD40 ligand, sargramostim, levamisol, vaccinia virus,Bacille Calmette-Guerin (BCG), liposomes, alum, Freund's complete orincomplete adjuvant, detoxified endotoxins, mineral oils, surface activesubstances such as lipolecithin, pluronic polyols, polyanions, peptides,and oil or hydrocarbon emulsions. Carriers for inducing a T cell immuneresponse which preferentially stimulate a cytolytic T cell responseversus an antibody response are preferred, although those that stimulateboth types of response can be used as well. In cases where the agent isa polypeptide, the polypeptide itself or a polynucleotide encoding thepolypeptide can be administered. The carrier can be a cell, such as anantigen presenting cell (APC) or a dendritic cell. Antigen presentingcells include such cell types aas macrophages, dendritic cells and Bcells. Other professional antigen-presenting cells include monocytes,marginal zone Kupffer cells, microglia, Langerhans' cells,interdigitating dendritic cells, follicular dendritic cells, and Tcells. Facultative antigen-presenting cells can also be used. Examplesof facultative antigen-presenting cells include astrocytes, follicularcells, endothelium and fibroblasts. The carrier can be a bacterial cellthat is transformed to express the polypeptide or to deliver apolynucleoteide which is subsequently expressed in cells of thevaccinated individual. Adjuvants, such as aluminum hydroxide or aluminumphosphate, can be added to increase the ability of the vaccine totrigger, enhance, or prolong an immune response. Additional materials,such as cytokines, chemokines, and bacterial nucleic acid sequences,like CpG, are also potential adjuvants. Other representative examples ofadjuvants include the synthetic adjuvant QS-21 comprising a homogeneoussaponin purified from the bark of Quillaja saponaria and Corynebacteriumparvu (McCune et al., Cancer, 1979; 43:1619). It will be understood thatthe adjuvant is subject to optimization. In other words, the skilledartisan can engage in routine experimentation to determine the bestadjuvant to use.

Further additives, such as preservatives, stabilizers, adjuvants,antibiotics, and other substances can be used as well. Preservatives,such as thimerosal or 2-phenoxy ethanol, can be added to slow or stopthe growth of bacteria or fungi resulting from inadvertentcontamination, especially as might occur with vaccine vials intended formultiple uses or doses. Stabilizers, such as lactose or monosodiumglutamate (MSG), can be added to stabilize the vaccine formulationagainst a variety of conditions, such as temperature variations or afreeze-drying process.

Viral vectors can be used to administer polynucleotides encoding apolypeptide comprising a mesothelin epitope. Such viral vectors includevaccinia virus and avian viruses, such as Newcastle disease virus.Others may be used as are known in the art.

One particular method for administering polypeptide vaccine is bypulsing the polypeptide onto an APC or dendritic cell in vitro. Thepolypeptide binds to MHC molecules on the surface of the APC ordendritic cell. Prior treatment of the APCs or dendritic cells withinterferon-γ can be used to increase the number of MHC molecules on theAPCs or dendritic cells. The pulsed cells can then be administered as acarrier for the polypeptide. Peptide pulsing is taught in Melero et al.,Gene Therapy 7:1167 (2000).

Naked DNA can be injected directly into the host to produce an immuneresponse. Such naked DNA vaccines may be injected intramuscularly intohuman muscle tissue, or through transdermal or intradermal delivery ofthe vaccine DNA, typically using biolistic-mediate gene transfer (i.e.,gene gun). Recent reviews describing the gene gun and muscle injectiondelivery strategies for DNA immunization include Tuting, Curr. Opin.Mol. Ther. (1999) 1: 216-25, Robinson, Int. J. Mol. Med. (1999) 4:549-55, and Mumper and Ledbur, Mol. Biotechnol. (2001) 19: 79-95. Otherpossible methods for delivering plasmid DNA includes electroporation andiontophoreses.

Another possible gene delivery system comprises ionic complexes formedbetween DNA and polycationic liposomes (see, e.g., Caplen et al. (1995)Nature Med. 1: 39). Held together by electrostatic interaction, thesecomplexes may dissociate because of the charge screening effect of thepolyelectrolytes in the biological fluid. A strongly basic lipidcomposition can stabilize the complex, but such lipids may be cytotoxic.Other possible methods for delivering DNA includes electroporation andiontophoreses.

The use of intracellular and intercellular targeting strategies in DNAvaccines may further enhance the mesothelin-specific antitumor effect.Previously, intracellular targeting strategies and intercellularspreading strategies have been used to enhance MHC class I or MHC classII presentation of antigen, resulting in potent CD8+ or CD4+ Tcell-mediated antitumor immunity, respectively. For example, MHC class Ipresentation of a model antigen, HPV-16 E7, was enhanced using linkageof Mycobacterium tuberculosis heat shock protein 70 (HSP70) (Chen, etal., (2000), Cancer Research, 60: 1035-1042), calreticulin (Cheng, etal., (2001) J Clin Invest, 108:669-678) or the translocation domain(domain II) of Pseudomonas aeruginosa exotoxin A (ETA(dII)) (Hung, etal., (2001) Cancer Research, 61: 3698-3703) to E7 in the context of aDNA vaccine. To enhance MHC class II antigen processing, the sortingsignals of the lysosome associated membrane protein (LAMP-1) have beenlinked to the E7 antigen, creating the Sig/E7/LAMP-1 chimera (Ji, et al,(1999), Human Gene Therapy, 10: 2727-2740). To enhance further thepotency of naked DNA vaccines, an intercellular strategy thatfacilitates the spread of antigen between cells can be used. Thisimproves the potency of DNA vaccines as has been shown using herpessimplex virus (HSV-1) VP22, an HSV-1 tegument protein that hasdemonstrated the remarkable property of intercellular transport and iscapable of distributing protein to many surrounding cells (Elliot, etal., (1997) Cell, 88: 223-233). Such enhanced intercellular spreading oflinked protein, results in enhancement of antigen-specific CD8+ Tcell-mediated immune responses and antitumor effect. Any such methodscan be used to enhance DNA vaccine potency against mesothlin-expressingtumors.

Mesothelin is known to be expressed in ovarian cancer, pancreaticcancer, mesothelioma, and squamous cell carcinomas carcinomas of theesophagus, lung, and cervix. Thus the vaccines of the invention areuseful for treating at least these types of tumors. Other tumors whichexpress mesothelin can also be treated similarly.

In one embodiment, the vaccines of the present invention comprise apolypeptide comprising at least one MHC Class I-binding epitope ofmesothelin or at least one MHC Class II-binding epitope of mesothelin.Alternatively, the vaccines of the present invention optionally comprisea polynucleotide encoding a polypeptide comprising at least one MHCClass I-binding epitope of mesothelin or at least one MHC ClassII-binding epitope of mesothelin. Optionally, the polypeptides of thevaccines (or the polypeptides encoded by the polynucleotides of thevaccines) comprise a plurality of MHC Class I-binding epitopes ofmesothelin and/or MHC Class II-binding epitopes of mesothelin. Themultiple epitopes of the polypeptides may bind the same or different MHCallelic molecules. In one embodiment, the epitopes of the polypeptidebind a diverse variety of MHC allelic molecules.

While MHC Class I-binding epitopes are effective in the practice of thepresent invention, MHC Class II-binding epitopes can also be used. Theformer are useful for activating CD8⁺ T cells and the latter foractivating CD4⁺ T cells. Publicly available algorithms can be used toselect epitopes that bind to MHC class I and/or class II molecules. Forexample, the predictive algorithm “BIMAS” ranks potential HLA bindingepitopes according to the predictive half-time disassociation ofpeptide/HLA complexes (23). The “SYFPEITHI” algorithm ranks peptidesaccording to a score that accounts for the presence of primary andsecondary HLA-binding anchor residues (25). Both computerized algorithmsscore candidate epitopes based on amino acid sequences within a givenprotein that have similar binding motifs to previously published HLAbinding epitopes. Other algorithms can also be used to identifycandidates for further biological testing.

Polypeptides for immunization to raise a cytolytic T cell response areoptionally from 8 to 25 amino acid residues in length. Although nonamersare specifically disclosed herein, any 8 contiguous amino acids of thenonamers can be used as well. The polypeptides can be fused to othersuch epitopic polypeptides, or they can be fused to carriers, such asB-7, interleukin-2, or interferon-γ. The fusion polypeptide can be madeby recombinant production or by chemical linkage, e.g., usingheterobifunctional linking reagents. Mixtures of polypeptides can beused. These can be mixtures of epitopes for a single allelic type of anMHC molecule, or mixtures of epitopes for a variety of allelic types.The polypeptides can also contain a repeated series of an epitopesequence or different epitope sequences in a series.

The effectiveness of an MHC Class I-binding epitope of mesothelin or anMHC Class II-binding epitope of mesothelin as an immunogen in a vaccinecan be evaluated by assessing whether a peptide comprising the epitopeis capable of activating T-lymphocytes from an individual having asuccessful immunological response to a tumor that overexpressesmesothelin (relative to normal tissue from which the tumor is derived),when the peptide is bound to an MHC molecule on an antigen-presentingcell and contacted with the T-lymphocytes under suitable conditions andfor a time sufficient to permit activation of T-lymphocytes. A specificexample of such an assessment is illustrated in Examples 1-4, below.

Multiple groups have cloned cDNAs encoding mesothelin, and the sequencesof the cDNA clones, as well as the sequence of the encoded mesothelinpolypeptides, have been reported in U.S. Pat. No. 6,153,430, Chang andPastan, Proc. Natl. Acad. Sci. USA, 93:136-140 (1996), Kojima et al., J.Biol. Chem., 270:21984-21990 (1995), and U.S. Pat. No. 5,723,318. Thesereferences, including the sequences of the mesothelin-encoding nucleicacids, corresponding mesothelin polypeptides, and fragments describedtherein, are incorporated by reference herein in their entirety.Mesothelin cDNA encodes a protein with a molecular weight ofapproximately 69 kD, i.e., the primary translation product. The 69 kDform of mesothelin is proteolytically processed to form a 40 kD maturemesothelin protein that is membrane-bound (Chang and Pastan (1996)). Theterm “mesothelin” as used herein encompasses all naturally occurringvariants of the mesothelin, regardless of the cell or tissue in whichthe protein is expressed, In one embodiment, the mesothelin proteincomprises one or more of the following amino acid sequences: SLLFLLFSL(SEQ ID NO:1); VLPLTVAEV (SEQ ID NO:2); ELAVALAQK (SEQ ID NO:3);ALQGGGPPY (SEQ ID NO:4); FYPGYLCSL (SEQ ID NO:5); and LYPKARLAF (SEQ IDNO:6). For instance, the mesothelin protein optionally comprises one,two, three, four, or five of these epitopes. In another embodiment, themesothelin protein comprises each of the following amino acid sequences:SLLFLLFSL (SEQ ID NO:1); VLPLTVAEV (SEQ ID NO:2); ELAVALAQK (SEQ IDNO:3); ALQGGGPPY (SEQ ID NO:4); FYPGYLCSL (SEQ ID NO:5); and LYPKARLAF(SEQ ID NO:6).

The vaccines of the invention optionally comprise mesothelin or apolynucleotide encoding mesothelin. For instance, the vaccine maycomprise or encode the mature form of mesothelin, the primarytranslation product, or the full-length translation product of themesothelin gene. In one embodiment, the vaccine comprises the cDNA ofmesothelin. In addition to the use of naturally occurring forms ofmesothelin (or polynucleotides encoding those forms), polypeptidescomprising fragments of mesothelin, or polynucleotides encodingfragments of mesothelin may be used in the vaccines. The polypeptides inthe vaccines or encoded by polynucleotides of the vaccines areoptionally at least about 95%, at least about 90%, at least about 85%,at least about 80%, at least about 75%, at least about 70%, at leastabout 65%, at least about 60%, at least about 55%, or at least about 50%identical to mesothelin.

In an alternative embodiment of the invention, the polypeptide of thevaccine or the polypeptide encoded by the polynucleotide of the vaccineis not a naturally-occurring mesothelin protein, such as the maturemesothelin protein, the primary translation product of mesothelin, orthe mature megakaryocyte potentiating factor.

In one embodiment, the MHC Class I-binding epitope of mesothelincomprises less than 15 amino acids, less than 14 amino acids, less than13 amino acids, less than 12 amino acids, or less than 11 amino acids inlength. In another embodiment, the MHC Class I-binding epitope ofmesothelin comprises at least seven or at least eight contiguous aminoacids present in a peptide selected from the group consisting ofSLLFLLFSL (SEQ ID NO:1), VLPLTVAEV (SEQ ID NO:2), ELAVALAQK (SEQ IDNO:3), ALQGGGPPY (SEQ ID NO:4), FYPGYLCSL (SEQ ID NO:5), and LYPKARLAF(SEQ ID NO:6). The MHC Class I-binding epitope of mesothelin is at least7 amino acids in length, at least 8 amino acids in length, or at least 9amino acids in length.

In addition, the MHC Class I-binding epitopes of mesothelin and the MHCClass II binding epitopes of mesothelin used in vaccines of the presentinvention need not necessarily be identical in sequence to the naturallyoccurring epitope sequences within mesothelin. The naturally occurringepitope sequences are not necessarily optimal peptides for stimulating aCTL response. See, for example, (Parkhurst, M. R. et al., J. Immunol.,157:2539-2548, (1996); Rosenberg, S. A. et al., Nat. Med., 4:321-327,(1998)). Thus, there can be utility in modifying an epitope, such thatit more readily induces a CTL response. Generally, epitopes may bemodified at two types of positions. The epitopes may be modified atamino acid residues that are predicted to interact with the MHCmolecule, in which case the goal is to create a peptide sequence thathas a higher affinity for the MHC molecule than does the parent epitope.The epitopes can also be modified at amino acid residues that arepredicted to interact with the T cell receptor on the CTL, in which casethe goal is to create an epitope that has a higher affinity for the Tcell receptor than does the parent epitope. Both of these types ofmodifications can result in a variant epitope that is related to aparent eptiope, but which is better able to induce a CTL response thanis the parent epitope.

Thus, the MHC Class I-binding epitopes of mesothelin identified in theExamples below (SEQ ID NO:1-6), or identified by application of themethods of the invention, and the MHC Class II-binding epitopes ofmesothelin identified by application of the methods of the invention canbe modified by the substitution of one or more residues at different,possibly selective, sites within the epitope sequence. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar,positively charged residues (His, Arg, Lys); Group 4—large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 4—large, aromaticresidues (Phe, Tyr, Trp). An acidic amino acid might also be substitutedby a different acidic amino acid or a basic (i.e., alkaline) amino acidby a different basic amino acid. Less conservative substitutions mightinvolve the replacement of one amino acid by another that has similarcharacteristics but is somewhat different in size, such as replacementof an alanine by an isoleucine residue.

Plasmids and viral vectors, for example, can be used to express a tumorantigen protein in a host cell. The host cell may be any prokaryotic oreukaryotic cell. Thus, for example, a nucleotide sequence derived fromthe cloning of mesothelin proteins, encoding all or a selected portionof the full-length protein, can be used to produce a recombinant form ofa mesothelin polypeptide via microbial or eukaryotic cellular processes.The coding sequence can be ligated into a vector and the loaded vectorcan be used to transform or transfect hosts, either eukaryotic (e.g.,yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells.Such techniques involve standard procedures which are well known in theart.

Typically, expression vectors used for expressing a polypeptide, in vivoor in vitro contain a nucleic acid encoding an antigen polypeptide,operably linked to at least one transcriptional regulatory sequence.Regulatory sequences are art-recognized and can be selected to directexpression of the subject proteins in the desired fashion (time andplace). Transcriptional regulatory sequences are described, for example,in Goeddel, Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990).

Suitable vectors for the expression of a polypeptide comprisingHLA-binding epitopes include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli. Mammalian expression vectors may contain bothprokaryotic and eukaryotic sequences in order to facilitate thepropagation of the vector in bacteria, and one or more eukaryotictranscription units that can be expressed in eukaryotic cells. ThepcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and selection inboth prokaryotic and eukaryotic cells. Alternatively, derivatives ofviruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus(pHEBo, pREP-derived and p205) can be used for transient expression ofproteins in eukaryotic cells. Vaccinia and avian virus vectors can alsobe used. The methods which may be employed in the preparation of vectorsand transformation of host organisms are well known in the art. Forother suitable expression systems for both prokaryotic and eukaryoticcells, as well as general recombinant procedures, see Molecular Cloning:A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

Other types of expression cassettes can also be used. For instance, thereferences described below in regard to viral, bacterial, and yeastvectors illustrate additional expression vectors which may be used inthe present invention.

In another embodiment of the invention, a polypeptide described herein,or a polynucleotide encoding the polypeptide, is delivered to a hostorganism in an immunogenic composition comprising yeast. The use of liveyeast DNA vaccine vectors for antigen delivery has been reviewedrecently and reported to be efficacious in a mouse model using wholerecombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1antigens (see Stubbs et al. (2001) Nature Medicine 7: 625-29).

The use of live yeast vaccine vectors is known in the art. Furthermore,U.S. Pat. No. 5,830,463, the contents of which are incorporated hereinby reference, describes particularly useful vectors and systems for usein the instant invention. The use of yeast delivery systems may beparticularly effective for use in the tumor/cancer vaccine methods andformulations of the invention as yeast appears to trigger cell-mediatedimmunity without the need for an additional adjuvant. Particularlypreferred yeast vaccine delivery systems are nonpathogenic yeastcarrying at least one recombinant expression system capable ofmodulating an immune response.

Bacteria can also be used as carriers for the epitopes of the presentinvention. Typically the bacteria used are mutant or recombinant. Thebacterium is optionally attenuated. For instance, a number of bacterialspecies have been developed for use as vaccines and can be used in thepresent invention, including, but not limited to, Shigella flexneri, E.coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonellatyphimurium, Salmonella typhi or mycobacterium. The bacterial vectorused in the immunogenic composition may be a facultative, intracellularbacterial vector. The bacterium may be used to deliver a polypeptidedescribed herein to antigen-presenting cells in the host organism. Theuse of live bacterial vaccine vectors for antigen delivery has beenreviewed recently (Medina and Guzman (2001) Vaccine 19: 1573-1580; Weissand Krusch, (2001) Biol. Chem. 382: 533-41; and Darji et al. (2000) FEMSImmunol and Medical Microbiology 27: 341-9). Furthermore, U.S. Pat. Nos.6,261,568 and 6,488,926, the contents of which are incorporated hereinby reference, describe systems useful for cancer vaccines.

Bacterially mediated gene transfer is particularly useful in geneticvaccination by intramuscular, intradermal, or oral administration ofplasmids; such vaccination leads to antigen expression in the vaccinee.Furthermore, bacteria can provide adjuvant effects and the ability totarget inductive sites of the immune system. Furthermore, bacterialvaccine vectors have almost unlimited coding capacity. The use ofbacterial carriers is often associated with still other significantbenefits, such as the possibility of direct mucosal or oral delivery.Other direct mucosal delivery systems (besides live viral or bacterialvaccine carriers) which can be used include mucosal adjuvants, viralparticles, ISCOMs, liposomes, and microparticles.

Both attenuated and commensal microorganisms have been successfully usedas carriers for vaccine antigens. Attenuated mucosal pathogens which maybe used in the invention include: L. monocytogenes, Salmonella spp., V.cholorae, Shigella spp., mycobacterium, Y. enterocolitica, and B.anthracis. Commensal strains which can be used in the invention include:S. gordonii, Lactobacillus spp., and Staphylococcus spp. The geneticbackground of the carrier strain used in the formulation, the type ofmutation selected to achieve attenuation, and the intrinsic propertiesof the immunogen can be adjusted to optimize the extent and quality ofthe immune response elicited. The general factors to be considered tooptimize the immune response stimulated by the bacterial carrierinclude: selection of the carrier; the specific background strain, theattenuating mutation and the level of attenuation; the stabilization ofthe attenuated phenotype and the establishment of the optimal dosage.Other antigen-related factors to consider include: intrinsic propertiesof the antigen; the expression system, antigen-display form andstabilization of the recombinant phenotype; co-expression of modulatingmolecules and vaccination schedules.

Salmonella typhimurium can be used as a bacterial vector in theimmunogenic compositions of the invention. Use of this bacterium as aneffective vector for a vaccine has been demonstrated in the art. Forinstance, the use of S. typhimurium as an attenuated vector for oralsomatic transgene vaccination has been described (see Darji et al.(1997) Cell 91: 765-775; and Darji et al. (2000) FEMS Immun and MedicalMicrobiology 27: 341-9). Indeed most knowledge of bacteria-mediated genetransfer has been acquired using attenuated S. typhimurium as carrier.Two metabolically attenuated strains that have been used include S.typhimurium aroA, which is unable to synthesize aromatic amino acids,and S. typhimurium 22-11, which is defective in purine metabolism.Several antigens have been expressed using these carriers: originally,listeriolysin and actA (two virulence factors of L. monocytogenes) andbeta-galactosidase (β-gal) of E. coli were successfully tested.Cytotoxic and helper T cells as well as specific antibodies could bedetected against these antigens following oral application of a singledose of the recombinant salmonella. In addition, immunization withSalmonella carrying a listeriolysin-encoding expression plasmid eliciteda protective response against a lethal challenge with L. monocytogenes.Oral transgene vaccination methodology has now been extended to includeprotective responses in herpes simplex virus 2 and hepatitis B infectionmodels, with cell-mediated immune responses detected at the mucosallevel.

In tumor models using β-gal as a surrogate tumor antigen, partialprotective immunity against an aggressive fibrosarcoma was induced byorally administering Salmonella carrying a 8-gal-encoding plasmid (seePaglia et al. (1998) Blood 92: 3172-76). In similar experiments using aβ-gal-expressing transfectant of the murine renal cell carcinoma lineRENCA, Zöller and Christ (Woo et al. (2001) Vaccine 19: 2945-2954)demonstrated superior efficacy when the antigen-encoding plasmid wasdelivered in bacterial carriers as opposed to using naked DNA.Interestingly, Salmonella can be used to induce a tumor growth retardingresponse against the murine melanoma B16; the Salmonella carry minigenesencoding epitopes of the autologous tumor antigens gp100 and TRP2 fusedto ubiquitin. This suggests that under such circumstances peripheraltolerance towards autologous antigens can be overcome. This wasconfirmed by the same group (Lode et al. (2000) Med Ped Oncol 35:641-646 using similar constructs of epitopes of tyrosine hydroxylase asautologous antigen in a murine neuroblastoma system. Furthermore, thesefindings were recently extended by immunizing mice that were transgenicfor human carcinogenic antigen (hCEA) using a plasmid encoding amembrane-bound form of complete hCEA. In this case, a hCEA-expressingcolon carcinoma system was tested and protection against a lethalchallenge with the tumor could be improved by systemic application ofinterleukin 2 (IL-2) as adjuvant during the effector phase (see Xiang etal. (2001) Clin Cancer Res 7: 856s-864s).

Another bacterial vector which may be used in the immunogeniccompositions described herein is Salmonella typhi. The S. typhi straincommonly used for immunization-Ty21a galE—lacks an essential componentfor cell-wall synthesis. Recently developed improved strains includethose attenuated by a mutation in guaBA, which encodes an essentialenzyme of the guanine biosynthesis pathway (Pasetti et al., Infect.Immun. (2002) 70:4009-18; Wang et al., Infect. Immun. (2001) 69:4734-41;Pasetti et al., Clin. Immunol. (1999) 92:76-89). Additional referencesdescribing the use of Salmonella typhi and/or other Salmonella strainsas delivery vectors for DNA vaccines include the following: Lundin,Infect. Immun. (2002) 70:5622-7; Devico et al., Vaccine, (2002)20:1968-74; Weiss et al., Biol. Chem. (2001) 382:533-41; and Bumann etal., FEMS Immunol. Med. Microbiol. (2000) 27:357-64.

The vaccines and immunogenic compositions of the present invention canemploy Shigella flexneri as a delivery vehicle. S. flexneri representsthe prototype of a bacterial DNA transfer vehicle as it escapes from thevacuole into the cytosol of the host cell. Several attenuated mutants ofS. flexneri have been used successfully to transfer DNA to cell lines invitro. Auxotrophic strains were defective in cell-wall synthesis(Sizemore et al. (1995) Science 270: 299-302 and Courvalin et al. (1995)C R Acad Sci Ser III, 318: 1207-12), synthesis of aromatic amino acids(Powell et al. (1996) Vaccines 96: Molecular Approaches to the Controlof Infectious Disease; Cold Spring Harbor Laboratory Press) or synthesisof guanine nucleotides (Anderson et al. (2000) Vaccine 18: 2193-2202).

The vaccines and immunogenic compositions of the present invention cancomprise Listeria monocytogenes (Portnoy et al, Journal of Cell Biology,158:409-414 (2002); Glomski et al., Journal of Cell Biology,156:1029-1038 (2002)). The ability of L. monocytogenes to serve as avaccine vector has been reviewed in Wesikirch, et al., Immunol. Rev.158:159-169 (1997). Strains of Listeria monocytogenes have recently beendeveloped as effective intracellular delivery vehicles of heterologousproteins providing delivery of antigens to the immune system to inducean immune response to clinical conditions that do not permit injectionof the disease-causing agent, such as cancer (U.S. Pat. No. 6,051,237;Gunn et al., J. Of Immunology, 167:6471-6479 (2001); Liau, et al.,Cancer Research, 62: 2287-2293 (2002); U.S. Pat. No. 6,099,848; WO99/25376; and WO 96/14087) and HIV (U.S. Pat. No. 5,830,702). Arecombinant L. monocytogenes vaccine expressing an lymphocyticchoriomeningitis virus (LCMV) antigen has also been shown to induceprotective cell-mediated immunity to the antigen (Shen et al., Proc.Natl. Acad. Sci. USA, 92: 3987-3991 (1995).

As a facultative intracellular bacterium, L. monocytogenes elicits bothhumoral and cell-mediated immune responses. Following entry of Listeriainto a cell of the host organism, the Listeria producesListeria-specific proteins that enable it to escape from thephagolysosome of the engulfing host cell into the cytosol of that cell.Here, L. monocytogenes proliferates, expressing proteins necessary forsurvival, but also expressing heterologous genes operably linked toListeria promoters. Presentation of peptides of these heterologousproteins on the surface of the engulfing cell by MHC proteins permit thedevelopment of a T cell response. Two integration vectors that areuseful for introducing heterologous genes into the bacteria for use asvaccines include pL1 and pL2 as described in Lauer et al., Journal ofBacteriology, 184: 4177-4186 (2002).

In addition, attenuated forms of L. monocytogenes useful in immunogeniccompositions have been produced. The ActA protein of L. monocytogenes issufficient to promote the actin recruitment and polymerization eventsresponsible for intracellular movement. A human safety study hasreported that oral administration of an actA/plcB-deleted attenuatedform of Listeria monocytogenes caused no serious sequelae in adults(Angelakopoulos et al., Infection and Immunity, 70:3592-3601 (2002)).Other types of attenuated forms of L. monocytogenes have also beendescribed (see, for example, WO 99/25376 and U.S. Pat. No. 6,099,848,which describe auxotrophic, attenuated strains of Listeria that expressheterologous antigens).

Yersinia enterocolitica is another intraceullular bacteria that canoptionally be used as a bacterial vector in immunogenic compositions ofthe present invention. The use of attenuated strains of Yersinienterocolitica as vaccine vectors is described in PCT Publication WO02/077249.

In further embodiments of the invention, the immunogenic compositions ofthe invention comprise mycobacterium, such as Bacillus Calmette-Guerin(BCG). The Bacillus of Calmette and Guerin has been used as a vaccinevector in mouse models (Gicquel et al., Dev. Biol. Stand 82:171-8(1994)). See also, Stover et al., Nature 351: 456-460 (1991).

Alternatively, viral vectors can be used. The viral vector willtypically comprise a highly attenuated, non-replicative virus. Viralvectors include, but are not limited to, DNA viral vectors such as thosebased on adenoviruses, herpes simplex virus, avian viruses, such asNewcastle disease virus, poxviruses such as vaccinia virus, andparvoviruses, including adeno-associated virus; and RNA viral vectors,including, but not limited to, the retroviral vectors. Vaccinia vectorsand methods useful in immunization protocols are described in U.S. Pat.No. 4,722,848. Retroviral vectors include murine leukemia virus, andlentiviruses such as human immunodeficiency virus. Naldini et al. (1996)Science 272:263-267. Replication-defective retroviral vectors harboringa polynucleotide of the invention as part of the retroviral genome canbe used. Such vectors have been described in detail. (Miller, et al.(1990) Mol. Cell. Biol. 10:4239; Kolberg, R. (1992) J. NIH Res. 4:43;Cornetta, et al. (1991) Hum. Gene Therapy 2:215).

Adenovirus and adeno-associated virus vectors useful in this inventionmay be produced according to methods already taught in the art. (See,e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter (1992) Current Opinionin Biotechnology 3:533-539; Muzcyzka (1992) Current Top. Microbiol.Immunol. 158:97-129; Gene Targeting: A Practical Approach (1992) ed. A.L. Joyner, Oxford University Press, NY). Several different approachesare feasible.

Alpha virus vectors, such as Venezuelan Equine Encephalitis (VEE) virus,Semliki Forest virus (SFV) and Sindbis virus vectors, can be used forefficient gene delivery. Replication-deficient vectors are available.Such vectors can be administered through any of a variety of means knownin the art, such as, for example, intranasally or intratumorally. SeeLundstrom, Curr. Gene Ther. 2001 1:19-29.

Additional references describing viral vectors which could be used inthe methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.)Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F.et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: GeneTransfer and Expression Protocols, Murray, E. (ed.), Humana Press,Clifton, N.J. (1991); Miller, et al. (1995) FASEB Journal 9:190-199,Schreier (1994) Pharmaceutica Acta Helvetiae 68:145-159; Schneider andFrench (1993) Circulation 88:1937-1942; Curiel, et al. (1992) Human GeneTherapy 3:147-154; WO 95/00655; WO 95/16772; WO 95/23867; WO 94/26914;WO 95/02697 (Jan. 26, 1995); and WO 95/25071.

In another form of vaccine, DNA is complexed with liposomes or ligandsthat often target cell surface receptors. The complex is useful in thatit helps protect DNA from degradation and helps target plasmid tospecific tissues. The complexes are typically injected intravenously orintramuscularly.

Polynucleotides used as vaccines can be used in a complex with acolloidal dispersion system. A colloidal system includes macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is alipid-complexed or liposome-formulated DNA. In the former approach,prior to formulation of DNA, e.g., with lipid, a plasmid containing atransgene bearing the desired DNA constructs may first be experimentallyoptimized for expression (e.g., inclusion of an intron in the 5′untranslated region and elimination of unnecessary sequences (Felgner,et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g., withvarious lipid or liposome materials, may then be effected using knownmethods and materials and delivered to the recipient mammal. See, e.g.,Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, AmJ Physiol 268; Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat.No. 5,679,647.

In addition, complex coacervation is a process of spontaneous phaseseparation that occurs when two oppositely charged polyelectrolytes aremixed in an aqueous solution. The electrostatic interaction between thetwo species of macromolecules results in the separation of a coacervate(polymer-rich phase) from the supernatant (polymer-poor phase). Thisphenomenon can be used to form microspheres and encapsulate a variety ofcompounds. The encapsulation process can be performed entirely inaqueous solution and at low temperatures, and has a good chance,therefore, of preserving the bioactivity of the encapsulant. Indeveloping an injectable controlled release system, the complexcoacervation of gelatin and chondroitin sulfate to encapsulate a numberof drugs and proteins has been exploited (see Truong, et al. (1995) DrugDelivery 2: 166) and cytokines have been encapsulated in thesemicrospheres for cancer vaccination (see Golumbek et al. (1993) CancerRes 53: 5841). Anti-inflammatory drugs have also been incorporated forintra-articular delivery to the joints for treating osteoarthritis(Brown et al. (1994) 331: 290). U.S. Pat. Nos. 6,193,970, 5,861,159 and5,759,582, describe compositions and methods of use of complexcoacervates for use as DNA vaccine delivery systems of the instantinvention. In particular, U.S. Pat. No. 6,475,995, the contents of whichare incorporated herein by reference, teaches DNA vaccine deliverysystems utilizing nanoparticle coacervates of nucleic acids andpolycations which serve as effective vaccines when administered orally.

Antibodies can be isolated which are specific for a particular MHC ClassI- of Class II binding epitope of mesothelin. These antibodies may bemonoclonal or polyclonal. They can be used, inter alia, for isolatingand purifying polypeptides for use as vaccines. T-cell lines that bindto an MHC class I or class II-peptide complex comprising a particularMHC Class I- of Class II binding epitope of mesothelin are useful forscreening for T cell adjuvants and immune response enhancers. Such celllines can be isolated from patients who have been immunized with amesothelin-containing vaccine and who have mounted an effective T cellresponse to mesothelin.

To test candidate cancer vaccines in the mouse model, the candidatevaccine containing the desired tumor antigen can be administered to apopulation of mice either before or after challenge with the tumor cellline of the invention. Thus the mouse model can be used to test for boththerapeutic and prophylactic effects. Vaccination with a candidatevaccine can be compared to control populations that are either notvaccinated, vaccinated with vehicle alone, or vaccinated with a vaccinethat expresses an irrelevant antigen. If the vaccine is a recombinantmicrobe, its relative efficacy can be compared to a population ofmicrobes in which the genome has not been modified to express theantigen. The effectiveness of candidate vaccine can be evaluated interms of effect on tumor or ascites volume or in terms of survivalrates. The tumor or ascites volume in mice vaccinated with candidatevaccine may be about 5%, about 10%, about 25%, about 50%, about 75%,about 90% or about 100% less than the tumor volume in mice that areeither not vaccinated or are vaccinated with vehicle or a vaccine thatexpresses an irrevelant antigen. The differential in tumor or ascitesvolume may be observed at least about 10, at least about 17, or at leastabout 24 days following the implantation of the tumor cells into themice. The median survival time in mice vaccinated with a nucleicacid-modified microbe may be, for example, at least about 2, at leastabout 5, at least about 7, or at least about 10 days longer than in micethat are either not vaccinated or are vaccinated with vehicle or avaccine that expresses an irrelevant antigen.

The mouse model can be used to test any kind of cancer treatment knownin the art. These may be conventional or complementary medicines. Thesecan be immunological agents or cytotoxic agents. For example, thecandidate cancer treatment may be radiation therapy, chemotherapy, orsurgery. The candidate cancer treatment may be a combination of two ormore therapies or prophylaxes, including but not limited to anti-canceragents, anti-tumor vaccines, radiation therapy, chemotherapies, andsurgery.

Any oncogene known in the art can be used to make the peritoneal ormesothelium cell line for making the mouse model. Such oncogenes includewithout limitation, Ki-ras, Erb-B2, N-ras, N-myc, L-myc, C-myc, ABL1,EGFR, Fos, Jun, c-Ha-ras, and SRC.

The vaccines, polynucleotides, polypeptides, cells, and viruses of thepresent invention can be administered to either human or other mammals.The other mammals can be domestic animals, such as goats, pigs, cows,horses, and sheep, or can be pets, such as dogs, rabbits, and cats. Theother mammals can be experimental subjects, such as mice, rats, rabbits,monkeys, or donkeys.

A reagent used in therapeutic methods of the invention is present in apharmaceutical composition. Pharmaceutical compositions typicallycomprise a pharmaceutically acceptable carrier, which meets industrystandards for sterility, isotonicity, stability, and non-pyrogenicityand which is nontoxic to the recipient at the dosages and concentrationsemployed. The particular carrier used depends on the type andconcentration of the therapeutic agent in the composition and theintended route of administration. If desired, a stabilizing compound canbe included. Formulation of pharmaceutical compositions is well knownand is described, for example, in U.S. Pat. Nos. 5,580,561 and5,891,725.

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to that amount of active ingredient that increases anti-tumorcytolytic T-cell activity relative to that which occurs in the absenceof the therapeutically effective dose.

For any substance, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model also can be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeuticallyeffective in 50% of the population) and LD50 (the dose lethal to 50% ofthe population), can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD50/ED50.

Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activeingredient or to maintain the desired effect. Factors that can be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions can be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc. Effective in vivo dosages of polynucleotidesand polypeptides are in the range of about 100 ng to about 200 ng, 500ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg,and about 20 μg to about 100 μg.

Desirable immunogens for use as anti-tumor vaccines are those which arehighly differentially expressed between tumors and their correspondingnormal tissues. Expression differences are preferably at least 2-fold,3-fold, 4-fold, 5-fold, or even 10 fold. Expression can be measured byany means known in the art, including but not limited to SAGE,microarrays, Northern blots, and Western blots. Interest in suchproteins as immunogens is enhanced by determining that humans respond toimmunization with the protein (or gene encoding it) by generating CD4 orCD8 T cells which are specifically activated by the protein. Testing forsuch activation can be done, inter alia, using TAP deficient cell linessuch as the human T2 cell line to present potential antigens in an MHCcomplex. Activation can be measured by any assay known in the art. Onesuch assay is the ELISPOT assay. See references 33-35.

Future responses to tumor vaccines can be predicted based on theresponse of CD8+ and or CD4+ T cells. If the tumor vaccine comprisesmesothelin or at least one T cell epitope of mesothelin, then monitoringof the of CD8+ and or CD4+ response to mesothelin provides usefulprognostic information. A robust CD8+ and or CD4+ response indicatesthat the patient has mounted an effective immunological response andwill survive significantly longer than those who have not mounted such aresponse. The tumor vaccine may comprise whole tumor cells, particularlypancreatic, ovarian or mesothelioma cells. The tumor vaccine maycomprise a polyethylene glycol fusion of tumor cells and dendriticcells. The tumor vaccine may comprise apoptotic or necrotic tumor cellswhich have been incubated with dendritic cells. The tumor vaccine maycomprise mRNA or whole RNA which has been incubated with dendriticcells. The T cell responses to mesothelin can be measured by any assayknown in the art, including an ELISPOT assay. Alternatively, futureresponse to such a tumor vaccine can be monitored by assaying for adelayed type hypersensitivity response to mesothelin. Such a responsehas been identified as a positive prognostic indicator.

Test substances which can be tested for use as a potential drug orimmune enhancing agent can be any substance known in the art. Thesubstance can be previously known for another purpose, or it can bepreviously unknown for any purpose. The substance can be a purifiedcompound, such as a single protein, nucleic acid, or small molecule, orit can be a mixture, such as an extract from a natural source. Thesubstance can be a natural product, or it can be a synthetic product.The substance can be specifically and purposefully synthesized for thispurpose or it can be a substance in a library of compounds which can bescreened.

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1

To identify genes that can serve as potential immune targets for themajority of pancreatic adenocarcinoma patients, we focused only on thosegenes that were non-mutated, overexpressed by the majority of pancreaticcancer patients, and overexpressed by the vaccine cell lines. One geneat the top of this list was mesothelin (20, 21). For comparison andvalidation purposes we also looked at prostate stem cell antigen (PSCA).SAGE data demonstrated PSCA to be expressed by pancreatic tumors atsimilar levels to that of mesothelin (22).

We used the combination of two public use computer algorithms (23-25) topredict peptide nonamers that bind to three common human leukocyteantigen (HLA)-class I molecules. All 14 patients treated with theallogeneic GM-CSF vaccine express at least one of these HLA-Class Imolecules (Table 2). The predictive algorithm “BIMAS”, ranks potentialHLA binding epitopes according to the predictive half-timedisassociation of peptide/HLA complexes (23). The “SYFPEITHI” algorithmranks peptides according to a score that accounts for the presence ofprimary and secondary HLA-binding anchor residues (25). Bothcomputerized algorithms score candidate epitopes based on amino acidsequences within a given protein that have similar binding motifs topreviously published HLA binding epitopes. We synthesized the top tworanking mesothelin epitopes for HLA-A2, HLA-A3, and HLA-A24 and the topsix PSCA epitopes for each MHC molecule favored by both algorithms(Table 1), since at least one of these three HLA class I molecules isexpressed by the 14 patients that were treated in our vaccine study(Table 2).

TABLE 1 Mesothelin peptides predicted to bind to HLA A2, A3, and A24.HLA- Amino Acid SEQ ID Amino Acid Restriction Sequence NO: Position inProtein HLA-A2 SLLFLLFSL 1 Mesothelin A2₍₂₀₋₂₈₎ HLA-A2 VLPLTVAEV 2Mesothelin A2₍₅₃₀₋₅₃₈₎ HLA-A2 LLALLMAGL 13 PSCA A2₍₅₋₁₃₎ HLA-A2ALQPGTALL 14 PSCA A2₍₁₄₋₂₂₎ HLA-A2 ALLPALGLL 15 PSCA A2₍₁₀₈₋₁₁₆₎ HLA-A3ELAVALAQK 3 Mesothelin A3₍₈₃₋₉₂₎ HLA-A3 ALQGGGPPY 4 MesothelinA3₍₂₂₅₋₂₃₄₎ HLA-A3 ALQPAAAIL 16 PSCA A3₍₉₉₋₁₀₇₎ HLA-A3 LLALLMAGL 13 PSCAA3₍₅₋₁₃₎ HLA-A3 ALQPGTALL 14 PSCA A3₍₁₄₋₂₂₎ HLA-A3 LLPALGLLL 17 PSCAA3₍₁₀₉₋₁₁₇₎ HLA-A3 QLGEQCWTA 18 PSCA A3₍₄₃₋₅₁₎ HLA-A3 ALLCYSCKA 19 PSCAA3₍₂₀₋₂₈₎ HLA-A24 FYPGYLCSL 5 Mesothelin A24₍₄₃₅₋₄₄₄₎ HLA-A24 LYPKARLAF6 Mesothelin A24₍₄₇₅₋₄₈₄₎ HLA-A24 DYYVGKKNI 20 PSCA A24₍₇₆₋₈₄₎ HLA-A24ALLPALGLL 15 PSCA A24₍₁₀₈₋₁₁₆₎ HLA-A24 ALQPAAAIL 16 PSCA A24₍₉₉₋₁₀₇₎HLA-A24 LLPALGLLL 17 PSCA A24₍₁₀₉₋₁₁₇₎ HLA-A24 YYVGKKNIT 21 PSCAA24₍₇₇₋₈₅₎

The three peptides, HIV-gag A2₇₇₋₈₅ (SLYNTVATL; SEQ ID NO: 7) (48),HIV-NEF A3₉₄₋₁₀₃ (QVPLRPMTYK; SEQ ID NO: 8) (49), and tyrosinaseA24₂₀₆₋₂₁₄ (AFLPWHRLF; SEQ ID NO: 9) (50), are previously publishedepitopes that were used as control peptides for HLA-A2, A3, and A24binding, respectively. The Mesothelin A1₃₀₉₋₃₁₈ binding epitope(EIDESLIFY; SEQ ID NO: 22) was used as a negative control peptide forall binding studies. The M1 peptide (GILGFVFTL; SEQ ID NO: 10)₅₈₋₆₆(Gotch et al 1988) was used as a positive control for all of the HLA-A2studies.

TABLE 2 Selected Characteristics of the 14 patients treated with anallogeneic GM-CSF secreting pancreatic tumor vaccine. Post-vaccineDisease- # of total HLA Class I increase in free Overall Disease StatusDose ×10⁷ vaccines Expression at DTH to Auto Survival Survival Pt # T(cm) LN Margins Cells received the A Locus¹ Tumor² (months) (months) 13.0 5/17 − 1 2 A1, A2  0 11  14 2 2.7 3/17 + 1 1 A2, A3  0 6 14 3 2.53/11 + 1 1 A1, A3  0 9 18 4 2.5 3/14 − 5 1 A2, A29 0 8 10 5 2.7 2/23 − 54 A3, A3  0 15  39 6 2.5 2/17 − 5 3 A2, A24 0 13  27 7 4.0 4/13 + 10 1A31, A24  0 16  18 8 2.7 5/18 + 10 2 A2, A3  252 mm 60+  60+ 9 1.2 2/11− 10 1 A3, A31 0 8 21 10 2.0 11/27  − 50 1 A3, A30 0 9 17 11 3.5 2/32 +50 1 A1, A3  N/A 9 13 12 2.5 2/11 − 50 1 A3, A33 N/A 11  13 13 3.0 2/14− 50 4 A3, A23 100 mm 60+  60+ 14 3.0 0/14 + 50 4 A1, A24 110 mm 60+ 60+ Abbreviations: Pt = patient, # = number, T = tumor size at surgery,LN = number of positive lymph nodes/total number of lymph nodes sampled,HLA = human leukocyte antigen, DTH = delayed type hypersensitivitytesting, Auto = autologous, N/A = not assessed due to unavailability ofDTH cell reagents, + = still alive and disease-free. ¹HLA typing wasperformed serologically and confirmed molecularly. ²Delayed typehypersensitivity reactions to autologous tumor cells was assessed usingunpassaged autologous tumor cells. 10⁶ autologous tumor cells wereplaced pre-vaccination, and at 28 days post-vaccination. Reported arethe post-vaccination change in the product of the perpendiculardiameters (measured in mm) of the observed induration at 48 hours aftercell placement.

Binding of these epitopes to their respective HLA class I molecule wastested by pulsing TAP deficient T2 cells that expressed thecorresponding HLA class I molecule (T2-A2, T2-A3, or T2-A24 cells). Asshown in FIG. 1A, pulsing of two mesothelin-derived epitopes predictedto bind to HLA-A2 allows for detection of HLA-A2 on the cell surface ofT2-A2 cells by flow cytometry following staining with the HLA class Ispecific antibody, W6/32. In contrast, unpulsed T2 cells or T2 cellspulsed with an mesothelin epitope predicted to bind to HLA-A1 do notstain with the same antibody. Binding of T2 cells pulsed with twocandidate mesothelin derived HLA-A3 and two candidate HLA-A24 epitopesare shown in FIG. 1B and FIG. 1C, respectively. A similar bindingexperiment was done with the PSCA derived peptides for HLA-A2, HLA-A3,and HLA-A24. (FIG. 1D, FIG. 1E and FIG. 1F).

Materials and Methods: Identification of candidate genes and epitopeselection. SAGE was used to identify mesothelin as one of the genesoverexpressed in pancreatic cancer cell lines and fresh tissue aspreviously reported (20, 21). Two computer algorithms that are availableto the general public and accessible through the internet were used topredict peptides that bind to HLA A2, A3, and A24 molecules. “BIMAS” wasdeveloped by K. C. Parker and collaborators http://bimas.dcrt.nih.gov/(NIH) that determined the optimal binding for the most common HLA classI molecule types (23). “SYFPEITHI” was developed by Rammensee et al. andranks the peptides according to a score that takes into account thepresence of primary and secondary MHC-binding anchor residueshttp://www.uni-tuebingen.de/uni/kxi (24).

Materials and Methods: Peptides and T2 cell lines. Peptides weresynthesized by Macromolecular Resources (Fort Collins, Colo.) accordingto published sequences: M1 peptide GILGFVFTL (SEQ ID NO: 10), derivedfrom influenza matrix protein (amino acid positions 58-66) (28),Mesothelin A2 peptides and PSCA A2 peptides listed in table 1 wereidentified using the available databases, HIV-gag A2 peptide SLYNTVATL(SEQ ID NO: 7) (amino acid positions 75-83) (29) contain an HLA-A2binding motif. Mesothelin A3 peptides and PSCA A3 peptides and HIV-NEFA3 peptide QVPLRPMTYK (SEQ ID NO: 8) (amino acid positions 94-103) (30)contain an HLA-A3 binding motif Mesothelin A24 peptides and PSCA A24peptides and Tyrosinase peptide AFLPWHRLF (SEQ ID NO: 9) (amino acidpositions 206-214) (31) contain an HLA-A24 binding motif Stock solutions(1 mg/ml) of each peptide were prepared in 10% DMSO (J T Baker,Phillippsburg, N.J.) and further diluted in cell culture medium to yielda final peptide concentration of 10 ng/ml for each assay. The control M1peptide was initially dissolved in 100% DMSO and further diluted in cellculture medium using the same stock and final concentrations. The T2cells are a human B and T lymphoblast hybrid that only express theHLA-A*0201 allele (26). The human T2 cell line is a TAP deficient cellline that fails to transport newly digested HLA class I binding epitopesfrom the cytosol into the endoplasmic reticulum where these epitopeswould normally bind to nascent HLA molecules and stabilize them forexpression on the cell surface (26). The T2-A3 are T2 cells geneticallymodified to express the HLA-A301 allele and were a gift from Dr. WalterStorkus (University of Pittsburgh) (32). T2-A24 are T2 cells geneticallymodified to express the HLA-A24 allele. The HLA-A24 gene was a gift fromDr. Paul Robbins (Surgery Branch, National Cancer Institute) (31). T2cells were grown in suspension culture in RPMI-1640 (Gibco, GrandIsland, N.Y.), 10% serum (Hyclone, Logan, Utah) supplemented with 200 μML-Glutamine (Gibco, Grand Island, N.Y.), 50 units-μg/ml Pen/Strep(Gibco), 1% NEAA (Gibco), and 1% Na-Pyruvate (Gibco).

Materials and Methods: Peptide/MHC binding Assays. T2 cells expressingthe HLA molecule of interest were resuspended in AimV serum free media(Gibco) to a concentration of 2.5×10⁵ cells/ml and pulsed with 100-200micrograms of peptide at room temperature overnight. Pulsing at roomtemperature allows for optimizing the number of empty HLA moleculesavailable for binding each epitope (30). The cells were washed andresuspended at 1×10⁵ cells/ml. Peptide binding was determined by FACS(Beckon Dickenson, San Jose, Calif.) analysis.

Example 2

To determine if mesothelin and PSCA are recognized by CD8+ T cells, wescreened antigen-pulsed T2 cells with CD8+ T cell enriched PBL frompatients that have received an allogeneic GM-CSF secreting pancreatictumor vaccine. We previously reported the association of in vivopost-vaccination delayed type hypersensitivity (DTH) responses toautologous tumor in three of eight patients receiving the highest twodoses of vaccine. These “DTH responders” (each of whom had poorprognostic indicators at the time of primary surgical resection (27) arethe only patients who remain clinically free of pancreatic cancer >4years after diagnosis ((27), Table 2). PBL obtained prior to vaccinationand 28 days after the first vaccination were initially analyzed. T2-A3cells pulsed with the two A3 binding epitopes were incubated overnightwith CD8+ T cell enriched lymphocytes isolated from the peripheral bloodof patient 10 (non-DTH responder who relapsed 9 months after diagnosis)and 13 (DTH responder who remains disease-free) and analyzed using agamma interferon (IFN-γ) ELISPOT assay. The ELISPOT assay was chosenbecause it requires relatively few lymphocytes, is among the mostsensitive in vitro assays for quantitating antigen-specific T cells, andcorrelates number of antigen-specific T cells with function (cytokineexpression) (33-35). The number of IFN-γ spots per 1×105 CD8+ positive Tcells detected in the peripheral blood of the two patients prior tovaccination and twenty-eight days following the first vaccination inresponse to the two HLA-A3 binding mesothelin peptides are shown in FIG.2A.

Induction of mesothelin-specific T cells was detected twenty-eight daysfollowing vaccination in patient 13 a DTH responder, but not in patient10, a non-DTH responder. Similarly, post-vaccination induction ofmesothelin-specific CD8+ T cells was observed in two other disease-freeDTH responders (patient 8 and patient 14), but not for two other non-DTHresponders when tested with T2-A2 and T2-A24 cells pulsed with the A2(FIG. 2B) and A24 (FIG. 2C) binding epitopes, respectively. A summary ofthe ELISPOT results analyzing all 14 patients treated with theallogeneic vaccine on this study for the induction ofmesothelin-specific CD8+ T cells following the first vaccination areshown in FIG. 2D. These data demonstrate that there is a directcorrelation between observed post-vaccination in vivo DTH responses toautologous tumor, long term disease-free survival, and post-vaccinationinduction of mesothelin-specific T cell responses in this clinicaltrial. Specifically, each of the three DTH responders demonstrated apost-vaccination induction in T cell response to every mesothelinpeptide that matched their respective HLA type, whereas only one ofeleven DTH non-responders had an increased post-vaccinationmesothelin-specific T cell response and only to a single peptide. Thus,the in vitro measurement of mesothelin-specific T cells responsesrepresents a new candidate in vitro immune marker for predicting whichpatients will respond to this vaccine therapy.

Materials and Methods: Peripheral blood lymphocytes (PBL) and donors.Peripheral blood (100 cc pre-vaccination and 28 days after eachvaccination) were obtained from all fourteen patients who received anallogeneic GM-CSF secreting pancreatic tumor vaccine as part of apreviously reported phase I vaccine study (27). Informed consent forbanking lymphocytes to be used for this antigen identification study wasobtained at the time of patient enrollment into the study. Pre andpost-vaccine PBL were isolated by density gradient centrifugation usingFicoll-Hypaque (Pharmacia, Uppsala, Sweden). Cells were washed twicewith serum free RPMI-1640. PBL were stored frozen at −180° C. in 90%AIM-V media containing 10% DMSO.

Materials and Methods: Enrichment of PBL for CD8+ T cells. CD8+ T cellswere isolated from thawed PBL using Magnetic Cell Sorting of HumanLeukocytes as per the manufacturers directions (MACS, Miltenyi Biotec,Auburn, Calif.). Cells were fluorescently stained with CD8-PE antibody(Becton Dickenson, San Jose, Calif.) to confirm that the positivepopulation contained CD8+ T cells and analyzed by flow cytometry. Thisprocedure consistently yielded >95% CD8+ T cell purity.

Materials and Methods: ELISPOT assay. Multiscreen ninety-six wellfiltration plates (Millipore, Bedford, Mass.) were coated overnight at4° C. with 60 μl/well of 10 μg/ml anti-hIFN-γ mouse monoclonal antibody(Mab) 1-D1K (Mabtech, Nacka, Sweden). Wells were then washed 3 timeseach with 1×PBS and blocked for 2 hours with T cell media. 1×105 T2cells pulsed with peptide (10 ng/ml) in 100 μl of T cell media wereincubated overnight with 1×10⁵ thawed PBL that are purified to selectCD8+ T cells in 100 μl T-cell media on the ELISPOT plates in replicatesof six. The plates were incubated overnight at 37° C. in 5% CO2. Cellswere removed from the ELISPOT plates by washing six times with PBS+0.05%Tween 20 (Sigma, St. Louis, Mo.). Wells were incubated for 2 hours at37° C. in 5% CO2 using 60 μl/well of 2 μg/ml biotinylated Mabanti-hIFNgamma 7-B6-1 (Mabtech, Nacka, Sweden). The avidin peroxidasecomplex (Vectastain ELITE ABC kit, Vetcor Laboratories, Burlingame,Calif.) was added after washing six times with PBS/Tween 0.05% at 100 μlper well and incubated for one hour at room temperature. AEC-substratesolution (3-amino-9-ethylcarbazole) was added at 100 μl/well andincubated for 4-12 minutes at room temperature. Color development wasstopped by washing with tap water. Plates were dried overnight at roomtemperature and colored spots were counted using an automated imagesystem ELISPOT reader (Axioplan2, Carl Zeiss Microimaging Inc.,Thornwood, N.Y.).

Example 3

The above data clearly demonstrate a correlation of in vivo DTH responseto autologous tumor and long term disease-free survival with thepost-vaccination induction of mesothelin-specific CD8+ T cell responses.It is possible, however, that this correlation represents generalizedimmune suppression (in the patients who failed to demonstratepost-vaccination DTH responses to their autologous tumor and who haddisease progression), rather than a vaccine specific induction of T cellresponses to mesothelin in the DTH responder patients who remaindisease-free. To demonstrate that the post-vaccination induction ofmesothelin-specific CD8+ T cells is tumor antigen-specific, we evaluatedeach HLA-A2 positive patient for T cell responses to the HLA-A2-bindinginfluenza matrix peptide, M1 (28). We chose the influenza M1 peptidebecause most patients on the vaccine study had received an influenzavaccine sometime prior to enrollment. As shown in FIG. 3, all HLA-A2positive patients demonstrated similar pre- and post-vaccination T cellresponses to the M1 peptide. Pre-vaccination responses ranged from 19 to50 IFN-γ spots per 105 total CD8+ T cells, and post-vaccinationresponses remained about the same in each patient (FIG. 3). A similarstudy was not done for HLA-A3 and A24 positive patients because thereare no published influenza M1 epitopes known to bind these HLAmolecules.

We evaluated the lymphocytes from the same 14 patients for thepost-vaccination induction of CD8+ T lymphocytes directed against asecond overexpressed antigen, PSCA. In contrast to mesothelin, PSCA didnot elicit an immune response in the 3 DTH responders. Again, wesynthesized the top two ranking epitopes for HLA-A2, HLA-A3, and HLA-A24favored by both algorithms and analyzed these according to the sameprotocols used in the mesothelin experiments. We did not see anypost-vaccination induction of PSCA-specific T cells in any of thepatients; therefore, we synthesized 4 additional PSCA peptides for eachHLA class I molecule to ensure that we had not missed the immunogenicepitope. Analysis of these peptides also failed to demonstrate apost-vaccination induction of PSCA-specific CD8+ T cell responses (FIGS.4A, 4B, and 4C, respectively). PSCA specific responses could not bedemonstrated in the eight non-responders as well (FIG. 4 d). This resultfurther supports our finding that mesothelin is a relevant pancreatictumor antigen because there were no vaccine induced immune responses toPSCA even though they are similarly overexpressed in pancreatic canceron SAGE analysis. In addition, the PSCA data demonstrate thatoverexpression of a protein in a tumor is insufficient to predict theprotein's utility as a vaccine target.

Flow cytometry analysis of mesothelin and PSCA expression by the twoallogeneic vaccine cell lines is shown in FIG. 5. Interestingly,mesothelin is expressed equally by both vaccine cell lines whereas PSCAis only expressed by one of the vaccine cell lines (Panc 6.03).

Materials and Methods: CD8+ M1 specific T cell lines. M1 specific T celllines were generated by repeated in vitro stimulation of HLA-A201positive PBL initially with irradiated autologous dendritic cellsfollowed by irradiated autologous Ebstein Barr Virus (EBV) transformed Bcells, both pulsed with the HLA-A201 restricted epitope. This line wasstimulated biweekly using autologous EBV cells that were pulsed with 10μg peptide/ml of their respective peptides at 37° C. for 2 hours, washedtwice with RPMI-1640, and irradiated with 10,000 rads. T cells werestimulated at a 1:2 T cell to EBV cell ratio in T cell media (RPMI-1640,10% human serum (pooled serum collected at the Johns HopkinsHemapheresis Unit) containing 200 μM L-Glutamine, 50 units-μg/mlPen/Strep, 1% NEAA, and 1% Na-Pyruvate) supplemented with 20 cetus unitsIL-2/well and 10 ng/well IL-7. This line was used a positive control Tcell line in all assays.

Materials and Methods: Flow cytometry. The expression of mesothelin andPSCA on the vaccine lines was evaluated by flow cytometry analysis. Thevaccine lines were washed twice and resuspended in “FACS” buffer (HBSSsupplemented with 1% PBS, 2% FBS, and 0.2% sodium azide), then stainedwith mouse monoclonal mesothelin (CAK1) (Signet Laboratories, Dedham,Mass.) or mouse monoclonal to PSCA (clone 1G8, obtained from R.E.R.)followed by FITC-labeled goat antimouse IgG (BD PharMingen, San Jose,Calif.) for flow analysis in a FACScan analyzer (BD ImmunocytometrySystems).

These data demonstrate that mesothelin-specific CD8+ T cells aredetected following a single vaccination with an allogeneic GM-CSFsecreting tumor vaccine in DTH responders but not in non-DTH responders.The patients treated on the reported vaccine study received an initialvaccination 8-10 weeks following pancreaticoduodenectomy and 4 weeksprior to receiving a six month course of adjuvant chemoradiation (27).Six of these patients remained disease-free at the end of the six monthsand received up to 3 more vaccinations given one month apart. RepeatELISPOT studies were performed on serial CD8+ T cell enriched PBLsamples from these six patients following multiple vaccine treatments toassess the effect of chemoradiation and multiple vaccinations onmesothelin-specific T cell responses.

As shown in FIG. 6, two of the three DTH responders demonstrateddecreased mesothelin-specific T cell responses following the secondvaccination. In both patients, mesothelin-specific T cell responsesreturned to levels achieved after the initial vaccination by the fourthvaccination. The suppressed mesothelin-specific T cell responses thatwere observed following the second vaccine are likely the result of thechemotherapy that each patient received between the first and secondvaccination. Interestingly, one of the three patients demonstratedsimilar mesothelin-specific T cell responses after the first and secondvaccination. This DTH responder only received two vaccines because shesubsequently developed a late autoimmune antibody mediated complicationattributed to the Mitomycin-C that required medical intervention andwithdrawal from the vaccine study. In contrast, repeated vaccinationfailed to induce mesothelin-specific T cell responses in those patientswho did not demonstrate an initial mesothelin-specific T cell responsefollowing the first vaccination (FIG. 6).

These data describing CD8⁺ T cell responses induced by an allogeneicGM-CSF-secreting pancreatic tumor vaccine support the followingconclusions. First, mesothelin can serve as an in vitro biomarker ofvaccine-induced immune responses that correlate with in vivo responsesin patients with pancreatic adenocarcinoma. Second, the recognition ofmesothelin and lack of recognition of another overexpressed geneproduct, PSCA, by uncultured post-vaccination CD8⁺ T cells from patientsthat demonstrated evidence of in vivo immune responses in associationwith clinical responses validates this antigen identification approachas a rapid functional genomic-based approach for identifying immunerelevant tumor targets of CD8⁺ T cells.

Mesothelin is a 40 kilodalton transmembrane glycoprotein member of themesothelin/megakaryocyte potentiating factor (MPF) family expressed bymost pancreatic adenocarcinomas (36), (37-39). It has also been reportedto be expressed by ovarian cancers, mesotheliomas, and some squamouscell carcinomas (37-39). Mesothelin is known to be attached to the cellmembrane by a glycosylphosphatidyl-inositol anchor and is postulated tofunction in cell adhesion (36). Mesothelin and other members of its genefamily exhibit limited normal tissue expression. It therefore meetsthree important criteria that strongly favor its potential use as animmunogen in the future development of antigen-based vaccines forpatients with pancreatic adenocarcinoma and other tumor types thatoverexpress mesothelin: it is widely shared by most pancreatic andovarian cancers, it has a limited expression in normal tissues, and itinduces CD8⁺ T cell responses following vaccination with tumor cellsthat express this antigen.

The identification of shared, biologically relevant tumor antigensprovides the opportunity to design antigen-based vaccines that have thepotential to be more efficient at inducing anti-tumor immunity thancurrent whole cell vaccines. In addition, scale up of recombinantantigen-based approaches is technically more feasible than currentlyemployed whole tumor cell vaccines. However, recombinant antigen-basedvaccines require identification of antigens that are both broadlyexpressed by patients and that are immunogenic. Until now, T cellscreening of cDNA libraries, antibody screening of phage displaylibraries, or the biochemical elution and purification of antigens boundto MHC have identified the majority of known tumor antigens, many ofwhich appear to derive from shared, non-mutated genes that are eitheroverexpressed by or reactivated in tumor cells relative to normal tissue(3-13). Unfortunately, this expanding list of tumor associated antigensrecognized by T cells is limited mostly to melanoma because of thetechnical difficulty of isolating and propagating T cell lines andclones from vaccinated patients with other types of cancer. The tumorantigen identification approach disclosed herein is feasible because itonly requires a database of differentially expressed genes within agiven tumor, and banked, uncultured bulk PBL from vaccinated patients.Therefore, this antigen identification approach is rapid and can begeneralized to most types of cancer. In addition, the use of unculturedlymphocytes rather than T cell lines and clones that have been in longterm culture provide the advantage of identifying new biologicallyrelevant immune targets.

PSCA is a second gene product that was found to be overexpressed in ourSAGE pancreatic gene expression database. In fact, PSCA was shown to beoverexpressed at higher levels than even mesothelin. However,post-vaccination PSCA specific T cell responses were not detected in theDTH responders and DTH non-responder patients. It is unclear at thistime why a GM-CSF secreting allogeneic vaccine induces T cell responsesto one overexpressed antigen and not to a second similarly overexpressedantigen. It is possible that these two antigens are differentlyprocessed and presented during the initial priming event (58).

In this study we also demonstrate that mesothelin-specific T cells canbe induced against at least six different peptides presented by threedifferent HLA-A locus alleles. This finding provides further supportthat mesothelin can serve as a shared antigen. In this study, thehighest ranking antigenic epitopes predicted to be the best HLA-A allelebinding epitopes based on their motif, bound to their respective HLAalleles and were also recognized by mesothelin-specific T cells. Reportsanalyzing other tumor antigens have found that the highest rankingepitopes do not necessarily correlate with optimal recognition by Tcells (25). We also performed the computer algorithms on two melanomaantigens, tyrosinase and MAGE 1, to determine how their published HLA-A2binding peptides rank by this method. We found that our HLA-A2 bindingmesothelin epitopes were given similar scores as the known tyrosinaseand MAGE 1 HLA-A2 binding epitopes. This was also true for the publishedHLA-A2 HIV GAG and HLA-A3 HIV NEF epitopes that were used as controlantigens in our analyses. Choosing epitopes that rank high by bothalgorithms appears to be an important predictor of the probability ofbinding to the respective HLA molecule.

We have developed a functional genomic approach that identified acandidate pancreatic tumor antigen. This approach to antigenidentification facilitates the identification of other human cancerantigens that are biologically relevant immune targets. The correlationof in vitro T cell responses with in vivo measures of response validatesthe biologic importance of this approach. This approach is rapid andfeasible and can easily be adapted to identify antigens expressed byother cancer types. This in turn, should accelerate the development ofrecombinant antigen-based vaccines for most human cancer treatment.

REFERENCES FOR BACKGROUND OF THE INVENTION AND FOR EXAMPLES 1-4

-   1. Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E.,    Hruban, R. H. et. al. Genome expression profiles in normal and    cancer cells. Science, 276: 1268-1272, (1997).-   2. Zhang, W., Laborde, P. M., Coombes, K. R., Berry, D. A. &    Hamilton, S. R. Cancer genomics: promises and complexities. Clin    Cancer Res, 7: 2159-2167, (2001).-   3. Boon, T., Coulie, P. G. & Van den Eynde, B. Tumor antigens    recognized by T cells. Immunol Today, 18: 267-268, (1997).-   4. Rosenberg, S. A. Progress in human tumour immunology and    immunotherapy. Nature, 411: 380-384, (2001).-   5. Rosenberg, S. A. Identification of cancer antigens: impact on    development of cancer immunotherapies. Cancer J Sci Am, 6:    S208-S217, (2000).-   6. Chen, Y. T. Cancer vaccine: identification of human tumor    antigens by SEREX. Cancer J Sci Am, 6: S208-S217, (2000).-   7. Nakatsura, T., Senju, S., Ito, M., Nishimurs, Y & Itoh, K.    Cellular and humoral immune response to a human pancreatic caner    antigen, coactosin-like protein, originally defined by the SEREX    method. Eur. J. Immunol, 32: 826-836, (2002).-   8. Scanlan, M. J., Welt, S., Gordon, C. M., Chen, Y-T., Gure, A. O.    et al. Cancer-related serological recognition of human colon cancer:    identification of potential diagnostic and immunotherapeutic    targets. Can Res., 62: 4041-4047, (2002).-   9. Monji, M., Senju, S., Nakatsura, T., Yamada, K., Sawatsubashi,    M., et al. Head and neck cancer antigens recognized by the humoral    immune system. Biochem Biophys Res Commun, 294: 734-741, (2002).-   10. Mashino, K., Sadanaga, N., Tanaka, F., Yamaguchi, H.,    Nagashima, H. et al. Expression of multiple cancer-testis antigen    genes in gastrointestinal and breast carcinomas. Br J cancer, 85:    713-720, (2001).-   11. Skipper, J. C., Gulden, P. H., Hendrickson, R. C., Harthun, N.,    Caldwell, J. A. et al. Mass-spectrometic evaluation of    HLA-A*0201-associated peptides identifies dominant naturally    processed forms of CTL epitopes from MART-1 and gp 100. Int J    Cancer, 82: 669-677, (1999).-   12. Hogan, K. T., Eisinger, D. P., Cupp, S. B., Lekstrom, K. J.,    Deacon, D. D. et al. The peptide recognized by HLA-A68.2 restricted,    squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes    is derived from a mutated elongation factor 2 gene. Cancer Res, 58:    5144-5150, (1998).-   13. Cox, A. L., Skipper, J., Chen, Y., Henderson, R. A.,    Darrow, T. L. et al. Identification of a peptide recognized by five    melanoma-specific human cytotoxic T cell lines. Science, 264:    716-719, (1994).-   14. Jager, D., Jager, E. & Knuth, A. Vaccination for malignant    melanoma: recent developments. Oncology, 60:1-7, (2001).-   15. Lockhart, K. J., Dong, H., Byrne, M. C., Follettie, M. T.,    Gallo, M. V. et al. Expression monitoring by hybridization to    high-density oligonucleotide arrays. Nat. Biotechnol, 14: 1675-1680,    (1996).-   16. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative    monitoring of gene expression patterns with a complementary DNA    microarray. Science, 270: 467-470, (1995).-   17. Velculescu, V. E., Zhang, L., Vogelstein, B., & Kinzler, K. W.    Serial analysis of gene expression. Science, 270: 484-487, (1995).-   18. Duggan, D. J., Bittner, M., Chen, Y., Meltzer, R. & Trent, J. M.    Expression profiling using cDNA microarrays. Nat Genet, 21: 10-14,    (1999).-   19. Khan, J., Bittner, M. L., Chen, Y., Meltzer, P. S. &    Trent, J. M. DNA microarray technology: the anticipated impact on    the study of human disease. Biochem Biophys Acta, 1423: M17-M28,    (1999).-   20. Argani, P., Rosty, C. Reiter, R. E., Wilenz, R. E.,    Murgesan, S. R. et al. Discovery of new markers of caner through    serial analysis of gene expression: prostate stem cell antigen is    overexpressed in pancreatic adenocarcinoma. Cancer Res, 61:    4320-4324, (2001).-   21. Argani, P., Iacobuzio-Donahue, C. Ryu, B., Rosty, C. Goggins, M.    et al. Mesothelin is overexpressed in the vast majority of ductal    adenocarcinomas of the pancreas: identification of a new pancreatic    cancer marker by serial analysis of gene expression (SAGE). Clin    Cancer Res, 3862: 3862-3868, (2001).-   22. Ryu, B., Jones, J., Blades, N. J., Parmigiani, G.,    Hollingsworth, M. A., et al. Relationships and differentially    expressed genes among pancreatic cancers examined by large-scale    serial analysis of gene expression. Cancer Res, 62: 819-826, (2002).-   23. Parker, K. C., Bednarek, M. A., & Coligan, J. E. Scheme for    ranking potential HLA-A2 binding peptides based on independent    binding of individual peptide side chains. J Immunol, 152: 163-175,    (1994).-   24. Rammensee, H.-H., Bachmann, J., Emmerich, N. P. N.,    Bachor, O. A. & Stevanovic, S. SYFPEITHI: database for MHC ligands    and peptide motifs. Immunogenetics, 50: 213-219, (1999).-   25. Lu, J. C., E. Use of two predictive algorithms of the world wide    web for the identification of tumor-reactive T-cell epitopes. Cancer    Res, 60: 5223-5227, (2000).-   26. Salter, R. D., Howell, D. N. & Cresswell, P. Genes regulating    HLA class I antigen expression in T-B lymphoblast hybrids.    Immunogenetics, 21: 235-246, (1985).-   27. Jaffee, E. M., Hruban, R. H., Biedrzycki, B., Laheru, D.,    Schepers, K. et al. Novel allogeneic granulocyte-macrophage    colony-stimulating factor-secreting tumor vaccine for pancreatic    cancer: phase I trial of safety and immune activation. J Clin. Onc,    19: 145-156, (2001).-   28. Bednarek, M. A., Sauma, S. Y., Gammon, M. C., Porter, G.,    Tamhanker, S. et al. The minimum peptide epitope from the influenza    matrixprotein. J Immunol, 147: 4047-4053, (1991).-   29. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H.,    McHeyzer-Williams, M. G. et al Phenotypic analysis of    antigen-specific T lymphocytes. Science, 274: 94-96, (1996).-   30. Propato, A., Schiaffella, E., Vicenzi, E. Francavilla, V.,    Baloni, L. et al. Spreading of HIV-specific CD8+ T-cell repertoire    in long-term nonprogressors and its role in the control of viral    load and disease activity. Human Immunol, 62: 561-576, (2001).-   31. Kang, X., Kawankami, Y., el-Gamil, M., Wang, R., Sakaguchi, K.    et al. Identification of a tyrosinase epitiope recognized by HLA-A24    restricted, tumor-infiltrating lymphocytes. J Immunol, 155:    1343-1348, (1995).-   32. Anderson, K. S., Alexander, J., Wei, M. & Cresswell, P.    Intracellular Transport of class I MHC molecules in antigen    processing mutant cell lines. J Immunol, 151: 3407-3419, (1993).-   33. Miyahira, Y., Kenichiro, M., Rodriguez, M., Rodriguez, J. R.,    Esteban, M. et al. Quantification of antigen specific CD8⁺ T cells    using an ELISPOT assay. J Immunol Methods, 181: 45-54, (1995).-   34. McCutcheon, M., Wehner, N., Wensky, A., Kushner, M., Doan, S. et    al. A sensitive ELISPOT assay to detect low-frequency human T    lymphocytes. J Immunol Methods, 210: 149-166, (1997).-   35. Schmittel, A., Keilholz, U. & Scheibenbogen, C. Evaluation of    the interferon-γ ELISPOT-assay for the quantification of peptide    specific T lymphocytes from peripheral blood. J Immunol Methods,    210: 167-174, (1997).-   36. Chang, K. P., I. Molecular cloning of mesothelin,    differentiation antigen present on mesothelium, mesotheliomas, and    ovarian cancers. Proc Natl Acad. Sci USA, 93: 136-140, (1996).-   37. Scholler, N., Fu, N., Yang, Y., Ye, Z., Goodman, G. E., et al.    Soluble member(s) of the mesothelin/megakaryocyte potentiating    factor family are detectable in sera from patients with ovarian    carcinoma. Proc Natl Acad. Sci USA, 96: 11531-11536, (1999).-   38. Chowdhury, P. S., VIner, J. L., Beers, R., Pastan, I. Isolation    of high-affinity stable single-chain Fv specific for mesothelin from    DNA-immunized mice by phage display and construction of recombinant    immunotoxin with anti-tumor activity. Proc Natl Acad. Sci. USA, 95:    669-674, (1998).-   39. Hassan, R., VIner, J. L., Wang, Q. C., Margulies, I.    Kreitman, R. J., & Pastan, I. Anti-tumor activity of K1-LysPE38QQR,    an immunotoxin targeting mesothelin, a cell surface antigen    overexpressed in ovarian cancer and malignant mesothelioma. J    Immunother., 23: 472-479, (2000).-   40. Chang, K. P., I. & Willingham, M. C. Isolation and    characterization of monaclonal antibody, K1, reactive with ovarian    cancers and normal mesothelium. Int J Cancer, 50: 373-381, (1992).-   41. Simons, J. W., Jaffee, E. M., Weber, C. E., Levitsky, H. I.,    Nelson, W. G., et al. Bioactivity of autologous irradiated renal    cell carcinoma vaccines generated by ex vivo granulocyte-macrophage    colony-stimulating factor gene transfer. Cancer Res, 57: 1537-1546,    (1997).-   42. Soiffer, R., Lynch, T., Mihm, M., Jung, K., Rhuda, C. et al    Vaccination with irradiated autologous melanoma cells engineered to    secrete human granulocyte-macrophage colony-stimulating factor    generates potent antitumor immunity in patients with metastatic    melanoma. Proc Natl Acad. Sci USA, 95: 13141-13146, (1998).-   43. Sokol, J. E. Measurement of delayed skin test responses. N Engl    J Med, 29: 501-503, (1995).-   44. McCune, C. S., O'Donnell, R. W., Marquis, D. M. &    Sahasrabudhe, D. M. Renal cell carcinoma treated by vaccines for    active specific immunotherapy: correlation of survival with skin    testing by autologous tumor cells. Cancer Immunol Immunother, 32:    62-66, (1990).-   45. Oren, M. E. H., R. B. Delayed cutaneous hypersensitivity    reactions to membrane extracts of human tumour cells. Clin Exp    Immunol, 9: 45-56, (1977).-   46. Hoover Jr., H. C., Surdyke, M., Dangel, R. B., Peters, L. C. &    Hanna Jr., M. G. Delayed cutaneous hypersensitivity to autologous    tumor cells in colorectal cancer patients immunized with an    autologous tumor cell: bacillus calmette-guerin vaccine. Cancer Res,    44: 1671-1676, (1984).-   47. Berd, D., Maguire Jr., H. C., McCue, P. & Mastrangelo, M. J.    Treatment of metastatic melanoma with an autologous tumor-cell    vaccine: clinical and immunologic results in 64 patients. J Clin    Oncol, 8: 1858-1867, (1990).-   48. Berd, D., Maguire Jr., H. C., & Mastrangelo, M. J. Induction of    cell-mediated immunity to autologous melanoma cells and regression    of metastases after treatment with a melanoma cell vaccine preceded    by cyclophoshamide. Cancer Res, 46: 2572-2577, (1986).-   49. Disis, M. L., Schiffman, K., Gooley, T. A., McNeel, D. G.,    Rinn, K. et al. Delayed-type hypersensitivity response is a    predictor of peripheral blood T-cell immunity after HER-2/neu    peptide immunization. Clin Cancer Res, 6: 1347-1350, (2000).-   50. Disis, M. L., Gooley, T. A., Rinn, K., Davis, D., Piepkorn, M.    et al. Generation of T-cell immunity to the HER-2/neu protein after    active immunization with HER-2/neu peptide-based vaccines. J Clin    Oncol, 20: 2624-2632, (2002).-   51. Knutson, K. L., Schiffman, K., Cheever, M. A. & Disis, M. L.    Immunization of cancer patients with a HER-2/neu, HLA-A2 peptide,    p369-377, results in short-lived peptide-specific immunity. Clin    Cancer Res, 8: 1014-1018, (2002).-   52. Gjertsen, M. K., Buanes, T., Rosseland, A. R., Bakka, A.,    Gladhaug, I. et al Intradermal ras peptide vaccination with    granulocyte-macrophage colony-stimulating factor as adjuvant:    Clinical and immunological responses in patients with pancreatic    adenocarcinoma. Int J Cancer, 92: 441-450, (2001).-   53. Pittet, M. J., Speiser, D. E., Lienard, D., Valmori, D.,    Guillaume, P. et al. Expansion and functional maturation of human    tumor antigen-specific CD8+ T cells after vaccination with antigenic    peptide. Clin Cancer Res, 7: 796s-803s, (2001).-   54. Abrams, S. I., Khleif, S. N., Bergmann-Leitner, E. S.,    Kantor, J. A., Chung, Y. et al. Generation of stable CD4⁺ and CD8⁺ T    cell lines from patients immunized with ras oncogene-derived    peptides reflecting codon 12 mutations. Cellular Immunol, 182:    137-151, (1997).-   55. DiFronzo, L. A., Gupta, R. K., Essner, R., Foshag, L. J.,    O'Day, S. J., et al. Enhanced humoral immune response correlates    with improved disease-free and overall survival in american joint    committee on cancer stage II melanoma patients receiving adjuvant    polyvalent vaccine. J Clin Oncol, 20: 3242-3248, (2002).-   56. Hodi, F. S., Schmollinger, J. C., Soiffer, R. J., Saliga, R.,    Lynch, T. et al. ATP6S1 elicits potent humoral responses associated    with immune-mediated tumor destruction. Proc Natl Acad. Sci USA, 99:    6919-6924, (2002).-   57. Moulton, H. M., Yoshihara, P. H., Mason, D. H., Iverson, P. L.,    & Triozzi, P. L. Active specific immunotherapy with a β-human    chorionic gonadotropin peptide vaccine in patients with metastatic    colorectal cancer: antibody response is associated with improved    survival. Clin Cancer Res, 8: 2044-2051, (2002).-   58. Kedl R. M., R. W. A., Hildeman D. A., Schaefer B., Mitchell T.,    Kappler J., Marrack P. T cells Compete for Access to Antigen-bearing    Antigen-presenting Cells. Journal Experimental Medicine, 192:    1105-1113, 2000.-   59. Ochsenbein, A. F., Sierro, S., Odermatt, B., Pericin, M.,    Karrer, U. et al. Roles of tumour localization, second signals, and    cross priming in cyotoxic T-cell induction. Nature, 411: 1058-1064,    (2001).-   60. Huang, A. Y., Bruce, A. T., Pardoll, D. M., Levitsky, H. I. In    vivo cross-priming of MHC class I-restricted antigens requires the    TAP transporter. Immunity, 4: 349-355, (1996).-   61. Huang, A. Y. C., Golumbek, P., Ahmadzeadeh, M., Jaffee, E. M.,    Pardoll, D. M., et al. Role of bone marrow-derived cells in    presenting MHC class I-restricted tumor antigens. Science, 264:    961-965, (1994).-   62. Norbury, C. C., Princiotta, M. F., Bacik, I., Brutkewicz, R. R.,    Wood, P. et al Multiple antigen-specific processing pathways for    activating naïve CD8⁺ T cells in vivo. J Immunol, 166: 4355-4362,    (2001).-   63. den Haan, J. M. B., M. J. Antigen presentation to CD8+ T cells:    cross priming in infectious diseases. Curr Opin Immunol, 13:    437-441, (2001).-   64. Kovacsovics-Bankowski, M., & Rock, K. L. A phagosome-to-cytosol    pathway for exogenous antigens presented on MHC class I molecules.    Science, 267: 243-246, (1995).-   65. Albert, M. L., Sauter, B. & Bhardwaj, N. Dendritic cells acquire    antigen from apoptotic cells induce class I-restricted CTLs. Nature,    392: 86-89, (1998).-   66. Berard, F., Blanco, P., Davoust, J., Neidhart-Berard, E. M.,    Nouri-Shirazi, M. et al. Cross-priming of naïve CD8 T cells against    melanoma antigens using dendritic cells loaded with killed    allogeneic melanoma cells. J Exp Med, 192: 1535-1544, (2000).-   67. Hoffmann, T. K., Meidenbauer, N., Dworacki, G., Kanaya, H. &    Whiteside, T. L. Generation of tumor-specific T lymphocytes by    cross-priming with human dendritic cells ingesting apoptotic tumor    cells. Cancer Res, 60: 3542-3549, (2001).-   68. John, M., Flam, M. & Palma, N. Ten-year results of    chemoradiation for anal cancer: focus on late morbidity. Int J    Radiat Oncl Biol Phys, 34: 65-69, (1996).

Example 5

Construction of Mouse Tumor Cells by Co-transformation with HPV-16 E6and E7 and Activated ras Oncogene. Primary peritoneal cells of C57BL/6mice were immortalized by HPV-16 E6 and E7 and then transformed withpEJB expressing activated human c-Ha-ras gene. This co-transformationproduced a tumorigenic cell line.

C57BL/6 mouse peritoneal cells were collected and washed with 1×HBSS.The primary single cell suspension was cultured in vitro in RPMI1640,supplemented with 10% fetal calf serum, 50 units/mlpenicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 2mM nonessential amino acids, and grown at 370 C with 5% CO2.Transduction of HPV-16 E6 and E7 genes into primary peritoneal cells wasperformed using the LXSN16E6E7 retroviral vector, kindly provided byDenise A. Galloway (Fred Hutchinson Cancer Research Center, Seattle,Wash.) (Halbert, et al., (1991) J Virol, 65:473-478). HPV-16 E6- andE7-containing LXSN16E6E7 was used to infect CRIP cells to generaterecombinant virus with a wide host range. Primary peritoneal cells wereimmortalized by transduction as described previously (Halbert, et al.,(1991) J Virol, 65:473-478). Following transduction, the retroviralsupernatant was removed, and cells were grown in G418 (0.4 mg/ml)culture medium for an additional 3 days to allow for integration andexpression of recombinant retroviral genes. The immortalized lung(E6+E7+) cells were then transduced with pVEJB expressing activatedhuman c-Ha-ras gene, kindly provided by Chi V. Dang (The John HopkinsHospital, Baltimore, Md.), and selected with G418 (0.4 mg/ml) andhygromycin (0.2 mg/ml).

Example 6

Characterization of Histological and Pathological Features of WF-3.5×104 WF-3 tumor cells were injected into C57BL/6 miceintraperitoneally. 4 weeks later, mice were sacrificed to examine theformation of ascites and tumors. Removed organs were fixed with 4%buffered formaldehyde and histological sections were made, followed byroutine hematoxylin-eosin staining. Slides were observed under a lightmicroscope.

Mice were injected with 5×104 WF-3 tumor cells intraperitoneally andsacrificed 4 weeks later. This cell line was capable of generatingascites in mice challenged with tumor cells intraperitoneally (see FIG.7A). Morphologically, WF-3 tumor cells showed a papillary architectureresembling serous tumors found in the human ovary/peritoneum (FIG. 1B).Furthermore, the tumor showed a high level of mitotic activity,pleiomorphic nuclei, abnormal mitosis, and a high nuclear/cytoplasmicratio, consistent with a highly malignant neoplasm (see FIG. 7C).

FIG. 7 shows the generation and characterization of an ascitogenicovarian tumor cell line (WF-3). WF-3 tumor cells were injected intoC57BL/6 mice intraperitoneally at a dose of 1×10⁵ cells/mouse. Mice wereeuthanized 4 weeks after tumor challenge (see FIG. 7A) Representativegross picture to demonstrate ascites formation in mice. Note: Micedeveloped significant ascites with an increase in abdominal girth 4weeks after tumor challenge. FIG. 7B shows hematoxylin and eosinstaining of the explanted tumors viewed at 100× magnification. Thetumors displayed a papillary configuration, morphologically consistentwith tumors derived from the peritoneum or ovaries. FIG. 7C shows tumorsviewed at 400× magnification. The inset displays the features of a WF-3tumor cell in greater detail.

Example 7

MHC Class I and Class II Presentation of WF-3 Tumor Cells. WF-3 tumorcells were harvested and prepared for flow cytometry analysis. Anti-H-2Kb/H-2 Db monoclonal antibody or anti-1-Ab monoclonal antibody was addedfor the detection of MHC class I and class II expression on WF-3 tumorcells.

WF-3 tumor cells were harvested, trypsinized, washed, and resuspended inFACScan buffer. Anti-H-2 Kb/H-2 Db monoclonal antibody (Clone 28-8-6,PharMingen, San Diego, Calif.) or anti-1-Ab monoclonal antibody (Clone25-9-17, PharMingen, San Diego, Calif.) was added and incubated for 30min on ice. After washing twice in FACScan buffer, FITC-conjugated goatanti-mouse antibody (Jackson ImmunoResearch Lab. Inc., West Grove, Pa.)was added and incubated for 20 min on ice. Samples were resuspended inFACScan buffer. Analysis was performed on a Becton Dickinson FACScanwith CELLQuest software (Becton Dickinson Immunocytometry System,Mountain View, Calif.).

Our data indicate that WF-3 is positive for MHC class I expression (FIG.8A) but negative for MHC class II expression (FIG. 8B). In particular,FIG. 8 shows the MHC class I and II presentation on WF-3 tumor cells.WF-3 tumor cells were harvested, trypsinized, washed, and resuspended inFACSCAN buffer. Anti-H-2 Kb/H-2 Db monoclonal antibody or anti-I-Abmonoclonal antibody was added, followed by flow cytometry analysis todetect MHC class I and class II expression on WF-3 tumor cells. FIG. 8Ashows WF-3 tumor cells which were positive for MHC class I presentation(thick line) compared to the MHC class I-negative control (thin line).FIG. 8B shows the WF-3 tumor cells which were negative for MHC class IIpresentation. The thin line indicates staining of the MHC classII-negative control.

Example 8

Determination of Minimal Tumor Dose of WF-3 Tumor Cells to Lead toFormation of Lethal Ascites. WF-3 tumor cells were injected into C57BL/6mice intraperitoneally at various doses (1×104, 5×104, 1×105, and 1×106cells/mouse). Mice were monitored twice a week for formation of ascitesand tumors and sacrificed after 90 days. For survival following tumorchallenge, mice were challenged intraperitoneally with various doses ofWF-3 (1×104, 5×104, 1×105, and 1×106 cells/mouse) and monitored fortheir survival after tumor challenge.

As shown in FIG. 9A, all of the mice injected with 5×104, 1×105, and1×106 cells intraperitoneally formed ascites within 30 days. Meanwhile,20% of mice injected with 1×104/mouse were tumor-free and withoutascites formation 90 days after tumor challenge. All of the miceinjected with a dose of 5×104 tumor cells or greater died within 50 daysof tumor challenge (FIG. 9B). These data suggest that WF-3 tumor cellsare able to lead to formation of ascites and solid tumors in theperitoneum of mice and eventually kill the injected mice at a certaintumor challenge dose.

Example 9

Mesothelin is Highly Expressed in the WF-3 Preclinical Ovarian CancerModel. We have performed microarray analysis (Incyte GenomicsCorporation, Palo Alto, Calif.) to characterize the gene expressionprofile of WF-3 compared to pre-WF0. The pre-WF0 cell line was generatedby immortalizing mouse primary peritoneal cells with a retroviral vectorcarrying HPV-16 E6 and E7 genes using a previously described method(Lin, et al., (1996) Cancer Research, 56:21-26). We have chosen pre-WF0as a reference cell line in order to identify genes in WF-3 that arerelevant to tumorigenicity in later stages of ovarian cancer. Table 4(below) summarizes highly expressed genes present in WF-3 relative topre-WF0. As shown in Table 4, below, mesothelin is among the top 10up-regulated genes in WF-3, suggesting that WF-3 may be a suitablepreclinical model for developing mesothelin-specific cancerimmunotherapy against ovarian cancer.

TABLE 4 Summary of Specifically Expressed Genes in WF-3 Antigen BalancedSequence differential Marker Accession # expression EGF-containingfibulin-like extracellular AI156278 6.5 matrix protein 1 MesothelinAA673869 3.8 alpha-2-HS-glycoprotein AI386037 3.3 Protein kinase,cGMP-dependent, type II AA771678 3.2 sema domain, immunoglobulin domainAA241390 3.2 (Ig), short basic domain, secreted (semaphorin) 3EAnkyrin-like repeat protein AA792499 2.8 RIKEN cDNA 1300019103 geneAA600596 2.6 Matrix gamma-carboxyglutamate (gla) W88093 2.4 proteinserine (or cysteine) proteinase inhibitor, AA727967 2.3 clade F (alpha-2antiplasmin, pigment epithelium Derived factor). member 1 RIKENcDNA1200011C15 gene AA608330 2.2

Example 10

Expression of Mesothelin mRNA and Protein in WF-3 Tumor Cells. Wefurther confirmed the expression of mesothelin by the WF-3 cell lineusing RT-PCR.

RNA was extracted from WF-3 tumor cells using RNAzol (Gibco BRL,Gaithersburg, Md.) according to the manufacturer's instructions. RNAconcentration was measured and 1 mg of total cellular RNA was reversetranscribed in a 20 ml volume using oligo(dT) as a primer andSuperscript reverse transcriptase (Gibco BRL). One ml of cDNA wasamplified by the PCR using a set of primers(5′-CCCGAATTCATGGCCTTGCCAACAGCTCGA-3′ and5′-TATGGATCCGCTCAGCCTTAAAGCTGGGAG-3′; SEQ ID NOS: 11 and 12,respectively). The primer was derived from the published murinemesothelin cDNA sequence (Kojima, et al., (1995) J Biol Chem,270:21984-21990). PCR was performed in a 50 ml reaction mixture with 250mM of each dNTP, 100 nM of primers, 5 ml of 10× buffer (New EnglandBiolabs, Berverly, Mass.), and 1 U of Vent DNA polymerase (New EnglandBiolabs) using 30 cycles (94° C., 1-min denaturation; 55° C., 1-minannealing; and 72° C., 2-min extension). The reaction mixture (10 mlsamples) was analyzed using agarose gel electrophoresis (1%) in TAEbuffer containing 0.2 mg/ml ethidium bromide.

Murine mesothelin protein shares about 65% similarity with humanmesothelin protein. As shown in FIG. 10, we were able to detect mRNAexpression of murine mesothelin in WF-3 tumor by RT-PCR (lane 2) but notin the control, B-16 tumor cells (lane 3). Western blot analysis wasperformed to determine expression of mesothelin protein in WF-3 tumorcells. Tumor cells were stained with anti-mesothelin mouse polyclonalantibodies. Results of the Western blot analysis confirmed that WF-3 waspositive for mesothelin protein while B16 melanoma cells weremesothelin-negative (data not shown). Thus, our results indicate thatWF-3 cells express mesothelin mRNA and protein.

FIG. 10 shows expression of murine mesothelin in WF-3 tumor cells asdemonstrated by RT-PCR with gel electrophoresis. Western blot analysiswas also performed to confirm expression (not shown). As shown in FIG.10, RT-PCR was performed using the Superscript One-Step RT-PCR Kit(Gibco, BRL) and a set of primers: 5′-CCCGAATTCATGGCCTTGCCAA-CAGCTCGA-3′and 5′-TATGGATCCGCTCA GCCTTAAAGCTGGGAG-3′ (SEQ ID NOS: 11 and 12,respectively). Western blot analysis was also used to demonstrate theexpression of mesothelin protein in WF-3 tumor cells. Tumor cells werestained with anti-mesothelin mouse polyclonal antibody followed byFITC-conjugated goat anti-mouse IgG secondary antibody (data not shown).

Example 11

Mesothelin DNA Cancer Vaccine Immunotherapy. Using the peritoneal tumormodel described above we demonstrated the ability of a DNA vaccineencoding mesothelin to generate mesothelin-specific cytotoxic Tlymphocyte responses and antitumor effects greater than empty plasmidDNA. These data indicate that a DNA tumor vaccine targeting mesothelincan be used in treating or controlling ovarian carcinomas and othercancers in which mesothelin is highly expressed.

Plasmid DNA Construction. With the availability of themesothelin-expressing tumor cell line, WF-3, we created DNA vaccinesencoding mesothelin to test their antitumor effect against WF-3 inC57BL/6 mice. We used a mammalian cell expression vector, pcDNA3, togenerate a DNA vaccine encoding murine full-length mesothelin protein(total length: 625 aa).

For construction of pcDNA3-mesothelin, a DNA fragment encodingmesothelin was first amplified from WF-3 extracted RNA and a set ofprimers (5′-CCCGAATTCATGGCCT-TGCCAACAGCTCGA-3′ and5′-TATGGATCCGCTCAGCCTTAAAGCTGGGAG-3′; SEQ ID NOS: 11 and 12,respectively) by RT-PCR using the Superscript One-Step RT-PCR Kit(Gibco, BRL) and cloned into the EcoRI/BamHI sites of pcDNA3. The primerwas derived from the published murine mesothelin cDNA sequence (11). Theaccuracy of DNA constructs was confirmed by DNA sequencing.

Vaccination with a DNA Vaccine Encoding Mesothelin Protein ProtectsAgainst Challenge with Mesothelin-Expressing Ovarian Tumors. We testedthe ability of this pcDNA3-mesothelin DNA vaccine to protect againsttumor challenge with WF-3 cells. Preparation of DNA-Coated GoldParticles and Gene Gun Particle-Mediated DNA vaccination using ahelium-driven gene gun (Bio-rad, Hercules, Calif.) was performedaccording to a previously described protocol (Chen, et al., (2000)Cancer Research, 60: 1035-1042. DNA-coated gold particles (1 mgDNA/bullet) were delivered to the shaved abdominal region of C57BL/6mice using a helium-driven gene gun (Bio-rad, Hercules, Calif.) with adischarge pressure of 400 p.s.i.

For the tumor protection experiment, mice (ten per group) werevaccinated intradermally with 2 mg of pcDNA3-mesothelin DNA. One weeklater, mice received a booster with the same dose. Mice were challengedone week after booster with a lethal injection of 5×104 WF-3 tumor cellsintraperitoneally. Mice were monitored for evidence of ascites formationby palpation and inspection twice a week; the mice were sacrificed atday 90. The percentage of ascites-free mice in each vaccination groupwas determined.

Our data indicated that pcDNA3-mesothelin generated a high degree ofprotection (60%) against WF-3 tumor challenge. Controls were vaccinatedwith pcDNA3 vector alone (0%) or were not vaccinated (0%). FIG. 11 showsin vivo tumor protection experiments against WF-3 tumor growth usingmesothelin-specific DNA vaccines.

Example 12

Vaccination with pcDNA3-mesothelin Generate Mesothelin-SpecificCytotoxic Immune Responses. CD8+ T lymphocytes are important effectorcells for mediating antitumor immunity. Cytotoxic T lymphocyte (CTL)assays were performed to determine the cytotoxic effect ofmesothelin-specific CD8+ T cells generated by the pcDNA3-mesothelin DNAvaccine. Splenocytes from vaccinated mice served as effector cells afterbeing cultured with cell lysates containing mesothelin protein. WF-3tumor cells served as target cells.

Generation of Mesothelin-Containing Cell Lysates from Transfected 293Db,Kb Cells. To generate mesothelin containing cell lysates to pulsesplenocytes for the CTL assays, a total of 20 mg of pcDNA3-mesothelin orempty plasmid DNA was transfected into 5×10⁶ Db,Kb cells withlipofectamine 2000 (Life Technologies) according to the manufacturer'sprotocol. The transfected 293 Db,Kb cells were collected 40-44 h aftertransfection, then treated with three cycles of freeze-thaw. The proteinconcentration was determined using the Bio-Rad protein assay (Bio-Rad,Hercules, Calif.) according to vendor's protocol. Cell lysatescontaining mesothelin were used to pulse splenocytes obtained from thevarious vaccinated mice as described below.

Cytotoxic T Lymphocyte (CTL) Assays. Cytolysis was determined byquantitative measurements of lactate dehydrogenase (LDH) using CytoTox96non-radioactive cytotoxicity assay kits (Promega, Madison, Wis.)according to the manufacturer's protocol. Briefly, splenocytes wereharvested from vaccinated mice (5 per group) and pooled 1 week after thelast vaccination. Splenocytes were pulsed with 20 mg of cell lysates ina total volume of 2 ml of RPMI 1640, supplemented with 10% (vol/vol)fetal bovine serum, 50 units/ml penicillin/streptomycin, 2 mML-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids in a24-well tissue culture plate for 6 days as effector cells. WF-3 tumorcells were used as target cells. WF-3 cells were mixed with splenocytesat various effector/target (E:T) ratios. After 5 hr incubation at 370 C,50 μl of the cultured media were collected to assess the amount of LDHin the cultured media according to the manufacturer's protocol. Thepercentage of lysis was calculated from the following equation:100×(A−B)/(C−D), where A is the reading of experimental-effector signalvalue, B is the effector spontaneous background signal value, C ismaximum signal value from target cells, D is the target spontaneousbackground signal value.

Statistical Analysis. Statistical determinations were made using theStudent's t-test. Two-sided P values are presented in all experiments,and significance was defined as P<0.05. No mice were excluded fromstatistical evaluations.

As shown in FIG. 12, vaccination with pcDNA3-mesothelin generated asignificant percentage of specific lysis compared to vaccination withpcDNA3 or no vaccination (P<0.001, one-way ANOVA). These resultsindicate that vaccination with pcDNA3-mesothelin DNA is capable ofgenerating mesothelin-specific T cell-mediated specific lysis of WF-3.

Cytotoxic T Lymphocyte (CTL) assays which demonstrate specific lysisinduced by vaccination with mesothelin-specific DNA vaccines. Mice (5per group) were immunized with various DNA vaccines intradermally. Micereceived a booster with the same dose one week later. Splenocytes frommice were pooled 14 days after vaccination. To perform the cytotoxicityassay, splenocytes were cultured with mesothelin protein for 6 days andused as effector cells. WF-3 tumor cells served as target cells. WF-3cells were mixed with splenocytes at various E:T ratios. Cytolysis wasdetermined by quantitative measurements of LDH. Note: ThepcDNA3-mesothelin DNA vaccine generated a significantly higherpercentage of specific lysis than the other DNA vaccines (P<0.001). Thedata presented in this figure are from one representative experiment oftwo performed.

Example 13

In this example, we utilize an attenuated strain of Salmonellatyphimurium as a vehicle for oral genetic immunization.PcDNA3.1/myc-His(−) vectors expressing a myc-tagged version ofmesothelin were constructed. Following immunization with the recombinantS. typhimurium aroA strain harboring the mesothelin expression vector,we are able to detect high levels of expression of the mesothelin/mycfusion protein using an anti-myc antibody by immunoassay. The S.typhimurium auxotrophic aroA strain SL7202 S. typhimurium 2337-65derivative hisG46, DEL407 [aroA::Tn10(Tc-s)]), is used as carrier forthese in vivo studies (see Darji et al. (1997) Cell 91: 761-775; Darjiet al. (2000) FEMS Immunology and Medical Microbiology 27: 341-9). ThisS. typhimurium-based mesothelin DNA vaccine delivery system is then usedto test whether this vaccine can protect ovarian cancer cells challengeusing our WF-3 tumor model system.

REFERENCES FOR EXAMPLES 5-13

-   1. Condon, C. et al., DNA-based immunization by in vivo transfection    of, dendritic cells. Nat Med, 2: 1122-1128, 1996.-   2. Ji, H., et al., Targeting BPV-16 E7 to the endosomal-lysosomal    compartment enhances the antitumor immunity of DNA vaccines against    murine HPV-16 E7-expressing tumors. Human Gene Therapy, 10:    2727-2740, 1999.-   3. Chen, C. H., Ji, H., Suh, K. W., Choti, M. A., Pardoll, D. M.,    and Wu, T. C. Gene gun-mediated DNA vaccination induces antitumor    immunity against human papillornavirus type 16 E7-expressing uterine    tumor-metastases in the liver and lungs. Gene Ther, 6: 1972-1981,    1999.-   4. Chen, C.-H., Suh, K. W., Ji, H., Choti, M. A., Pardoll, D. M.,    and Wu, T.-C Antigen-specific immunotherapy for HPV-16 E7-expressing    tumors grown in liver. J. Hepatology, 33(1):91-8, 2000.-   5. Chen, C.-H., Wang, T.-L., Hung, C.-F., Yang, Y., Young, R. A.,    Pardoll, D. M., and Wu, T.-C. Enhancement of DNA vaccine potency by    linkage of antigen gene to an HSP70 gene. Cancer Research, 60:    1035-1042, 2000.-   6. Hung, C.-F., Cheng, W.-F., Hsu, K.-F., Chai, C.-Y., He, L., Ling,    M., and Wu, T.-C. Cancer immunotherapy using a DNA vaccine encoding    the translocation domain of a bacterial toxin linked to a tumor    antigen. Cancer Research, 61: 3698-3703, 2001.-   7. Hung, C.-F., Cheng, W.-F., Chai, C.-Y., Hsu, K.-F., He, L., Ling,    M., and Wu, T.-C. Improving vaccine potency through intercellular    spreading and enhanced NMC class I presentation of antigen. J    Immunol, 166: 5733-5740, 2001.-   8. Hung, C.-F., Hsu, K.-F., Cheng, W.-F., Chai, C.-Y., He, L., Ling,    M., and Wu, T.-C. Enhancement of DNA vaccine potency by linkage of    antigen gene to a gene encoding the extracellular domain of    Flt3-ligand. Cancer Research, 61: 1080-1088, 2001.-   9. Cheng, W. F., Hung, C. F., Chai, C. Y., Hsu, K. F., He, L., Ling,    M., and Wu, T. C. Tumor-specific immunity and antiangiogenesis    generated by a DNA vaccine encoding calreticulin linked to a tumor    antigen. J Clin Invest, 108: 669-678, 2001.-   10. Halbert, C. L., Demers, G. W., and Galloway, D. A. The E7 gene    of human papillomavirus type 16 is sufficient for immortalization of    human epithelial cells. J Virol, 65:473-478, 1991.-   11. Kojima, T., Oh-eda, M., Hattori, K., Taniguchi, Y., Tamura, M.,    Ochi, N., Yamaguchi, N. Molecular cloning and expression of    megakaryocyte potentiating factor cDNA. J Biol Chem, 270:    21984-21990, 1995.-   12. Lin, K.-Y., Guarnieri, F. G., Staveley-OCarroll, K. F.,    Levitsky, H. I., August, T., Pardoll, D. M., and Wu, T.-C. Treatment    of established tumors with a novel vaccine that enhances major    histocompatibility class II presentation of tumor antigen. Cancer    Research, 56: 21-26, 1996.-   13. Yokoyama, Y., Dhanabal, M., Griffloen, A. W., Sukhatme, V. P.,    and Ramakrishnan, S. Synergy between angiostatin and endostatin:    inhibition of ovarian cancer growth. Cancer Res, 60: 2190-2196,    2000.-   14. Huang, S., Robinson, 1. B., Deguan, A., Bucana, C. D., and    Fidler, I. J. Blockade of nuclear factor-kappaβ signaling inhibits    angiogenesis and tumorigenicity of human ovarian cancer cells by    suppressing expression of vascular endothelial growth factor and    interleukin 8. Cancer Res, 60: 5334 5339, 2000-   15. Nielsen, L. L., Shi, B., Hajian, G., Yaremko, B., Lipari, P.,    Ferrari, E., Gumani, M., Malkowski, M., Chen, J., Bishop, W. R., and    Liu, M. Combination therapy with the farnesyl protein transferase    inhibitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical    cancer models. Cancer Res, 59: 5896-5901, 1999.-   16. Mesiano, S., Ferrara, N., and Jaffe, R. B. Role of vascular    endothelial growth factor in ovarian cancer: inhibition of ascites    formation by immunoneutralization. Am J Pathol, 153: 1249-1256,    1998.-   17. Elliott G. and O'Hare, P. Intercellular trafficking and protein    delivery by a herpesvirus structural protein. Cell, 88: 223-233,    1997.-   18. Rosenberg, S. A. A new era for cancer immunotherapy based on the    genes that encodecancer antigens. Immunity, 10: 281-287, 1999.-   19. Boon, T., Cerottini, J. C., Van den Eynde, B., van der Bruggen,    P., and Van Pel, A. Tumor antigens recognized by T lymphocytes. Annu    Rev Immunol, 12: 337-365, 1994.-   20. Schena, M., Shalon, D, Davis, R. W., and Brown, P. O.    Quantitative monitoring of gene expression patterns with a    complementary DNA microarray. Science, 270: 467-470, 1995.-   21. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W.    Serial analysis of gene expression. Science, 270: 484-487, 1995.-   22. Lander, E. S., et al., Initial sequencing and analysis of the    human genome. Nature, 409: 860-921, 2001.-   23. Chen, Y. T., Scanlan, M. J., Sahin, U., Tureci, O., Gure, A. O,    Tsang, S., Williamson, B., Stockert, E., Pfreundschuh, M., and    Old, L. J. A testicular antigen aberrantly expressed in human    cancers detected by autologous antibody screening. Proc Natl Acad    Sci USA, 94: 1914-1918, 1997.-   24. Tureci, O., Sahin, U., Schobert, I., Koslowski, M., Scmitt, H.,    Schild, H. J., Stenner, F., Seitz, G., Ranunensee, H. G., and    Pfreundschuh, M. The SSX-2 gene, which is involved in the t(X; 18)    translocation of synovial sarcomas, codes for the human tumor    antigen HOM-MEL-40. Cancer Res, 56: 4766 4772, 1996.

We claim:
 1. A method of predicting future response to a tumor vaccinecomprising mesothelin or a peptide comprising at least one mesothelinMHC class I- or class II-binding epitope in a patient who has receivedthe vaccine, wherein the patient has a mesothelin-expressing tumor,comprising: testing the patient to determine if the patient has adelayed type hypersensitivity (DTH) response to mesothelin or thepeptide, wherein the presence of said response predicts a longersurvival time than the absence of said response.
 2. The method of claim1 wherein the vaccine comprises whole tumor cells.
 3. The method ofclaim 1 wherein the vaccine comprises pancreatic tumor cells.
 4. Themethod of claim 1 wherein the vaccine comprises ovarian tumor cells. 5.The method of claim 1 wherein the vaccine comprises mesothelioma cells.6. The method of claim 1 wherein the vaccine comprises mesothelin. 7.The method of claim 1 wherein the vaccine comprises the peptide.
 8. Themethod of claim 1 wherein the vaccine comprises the peptide comprisingat least one MHC class I-binding epitope of mesothelin.
 9. The method ofclaim 1 wherein the patient is tested for a DTH response to mesothelin.10. The method of claim 1 wherein the patient is tested for a DTHresponse to the peptide.
 11. The method of claim 1 wherein the patientis tested for a DTH response to the peptide comprising at least one MHCclass I-binding epitope of mesothelin.
 12. The method of claim 1 whereinthe patient has a pancreatic tumor.
 13. The method of claim 1 whereinthe patient has an ovarian tumor.
 14. The method of claim 1 wherein thepatient has a mesothelioma.
 15. The method of claim 1 wherein thevaccine comprises a bacterium.
 16. The method of claim 1 wherein thevaccine comprises Listerium monocytogenes.
 17. The method of claim 8wherein the vaccine comprises a bacterium.
 18. The method of claim 8wherein the vaccine comprises Listerium monocytogenes.
 19. A method ofpredicting future response to a tumor vaccine in a patient who hasreceived the vaccine, wherein the vaccine comprises a polynucleotideencoding a polypeptide comprising an MHC class I-binding epitope ofmesothelin or an MHC class II-binding epitope of mesothelin, comprising:testing the patient to determine if the patient has a delayed typehypersensitivity (DTH) response to mesothelin, wherein the presence ofsaid response predicts a longer survival time than the absence of saidresponse.
 20. The method of claim 19 wherein the vaccine comprises wholetumor cells.
 21. The method of claim 19 wherein the vaccine comprisespancreatic tumor cells.
 22. The method of claim 19 wherein the vaccinecomprises ovarian tumor cells.
 23. The method of claim 19 wherein thevaccine comprises mesothelioma cells.